Enantioselective oligomerization of alpha-hydroxy carboxylic acids and alpha-amino acids

ABSTRACT

An enzymatic synthesis and composition of oligomers and co-oligomers comprised of α-hydroxy carboxylic acids and α-amino acids or peptides is disclosed. In a preferred embodiment, a α-hydroxy carboxylic acid with a specific chiral configuration is linked by an amide linkage to a α-amino acid specific with a specific chiral configuration or linked by an amide linkage to a peptide made up of α-amino acid monomers having identical chiral configurations. Proteolytic enzymes catalyze oligomerization of the α-hydroxy carboxylic acid and α-amino acid. The degree and distribution of oligomerization varies upon the type and concentrations of different reaction mixtures utilized and upon the length of allowed reaction time. The resultant oligomers may be provided to animals such as ruminants as bioavailable amino acid supplements that are resistant to degradation in the rumen and other animals such as swine, poultry and aquatic animals.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/136,974, filed May 2, 2002, which claims priority from U.S.Provisional Application Serial No. 60/288,196, filed May 2, 2001, and asa continuation-in-part of U.S. patent application Ser. No. 09/699,946,filed Oct. 30, 2000, which claims priority from U.S. ProvisionalApplication Serial No. 60/162,725, filed Oct. 29, 1999 (now abandoned).The entire texts of U.S. Provisional Application Serial No. 60/288,196,U.S. patent application Ser. Nos. 09/699,946 and 10/136,974, and U.S.Provisional Application Serial No. 60/288,196 are hereby incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a process for the enantioselectivepreparation of oligomers consisting of α-amino acid isomers andco-oligomers consisting of α-hydroxy carboxylic acid isomers and α-aminoacid isomers. The present invention also relates to compositionscontaining such oligomers and co-oligomers and methods of use thereof.

In an effort to improve nutrition, the diets of ruminant animals havebeen supplemented with proteins and naturally occurring α-amino acids.Unfortunately, these proteins and α-amino acids can be subjected toextensive degradation in the rumen by ruminal microorganisms, therebyrendering the protein or amino acid unavailable to the animal forabsorption. This is not a very efficient utilization of the feed, whichis especially problematic in animals having increased nutritionalrequirements such as lactating dairy cows and fast growing animals suchas beef cattle.

One approach to solving this problem has been to modify or protect thedietary protein or amino acid by a variety of chemical and physicalmethods so that it escapes degradation in the rumen. For example,heating soybean meal has shown some promise in producing protectedproteins. However, the results were highly variable. Underheating theprotein resulted in no protection while overheating the protein resultedin the degradation of important essential amino acids. See, for example,Plegge, S. D., Berger, L. L. and Fahey Jr. G. C. 1982. Effect ofRoasting on Utilization of Soybean Meal by Ruminants. J. Anim. Sci.55:395 and Faldet, M. A., Son, Y. S. and Satter, L. D. 1992. Chemical,in vitro and in vivo evaluation of soybean heat-treated by variousprocessing methods. J. Dairy Sci. 75:789. Similarly, physical coating ofproteins with materials such as fats and calcium soaps of fats has beenmet with mixed success.

Therefore, there is a need to somehow protect the protein fromdegradation in the rumen in order to make it available to the animal inthe intestine where it can be properly absorbed. This would allow theanimal to get increased nutritional benefit from the feed. Increasingthe nutritional benefit of the feed can reduce the amount of feedrequired by the animals.

Dietary supplements such as proteins, naturally occurring α-amino acids,vitamins, minerals, and other nutrients are also used in aquaculture,(i.e., the cultivation of aquatic animals such as fish and crustaceans).Many of such supplements are difficult to provide, however, due to beingsoluble in water which causes the supplements to dissolve before theycan be ingested. Dietary supplements for use in aquaculture thereforeare preferably in an insoluble form in order to be ingested.

The role played by short chain peptides and their derivatives in theareas of nutrition science, flavor chemistry, and pharmacology hasprimed the advances in peptide chemistry. The inherent advantages ofenzymatic peptide synthesis has led to its evolution as an alternativeto chemical coupling methods (Fruton, J. S., 1992, Adv. Enzymology, 53,239-306). The thiol-protease papain is reported to be the most efficientcatalyst for aqueous phase synthesis of homo-oligomers of hydrophobicamino acids like leucine, methionine, phenylalanine, and tyrosine (A.Ferjancic, A. Puigserver and H. Gaertner, Biotech. Lett, 13(3) (1991)161-166). The equilibria of such reactions is tilted in favor ofsynthesis by the precipitation of hydrophobic oligomers. However, thedifficulty involved in the analysis of higher order, water insolubleoligomers, presents a unique challenge to biochromatography.

SUMMARY OF THE INVENTION

Among the objects of the present invention, therefore, is the provisionof an oligomer which is protected from degradation in the rumen of aruminant, the provision of such an oligomer which provides nutritionalor pharmacological benefit to the animal, and the provision of a processfor the preparation of such oligomers.

A further object of the present invention is the provision of aco-oligomer and oligomer that provides nutritional or pharmacologicalbenefit to animals, and the provision of a process for the preparationof such co-oligomers and oligomers.

A further object of the invention is the provision of a co-oligomeric oroligomeric coating for vitamins, minerals, or nutrients.

Another object of the present invention is the provision of a method topurify enantiomeric mixtures of α-hydroxy carboxylic acids, α-aminoacids, or combinations thereof.

Briefly, therefore, the present invention is directed to a process forthe preparation of an oligomer consisting of α-amino acid isomers. Theprocess comprises forming a reaction mixture containing (i) an enzymeand (ii) an an enantiomeric mixture of α-amino acid, or derivativethereof. An oligomer is formed that incorporates one enantiomer of theenantiomeric mixture of the α-amino acid or derivative thereof inpreference to the other enantiomer.

The present invention is further directed to a composition comprising aresidue of an α-hydroxy carboxylic acid bonded to a peptide by an amideor an ester linkage, said peptide comprising two or more α-amino acidresidues, each of said α-amino acids being independently selected fromthe group consisting of α-amino acids. Preferably, more than 50% of theα-amino acid residues in the peptide are of identical chirality.

The present invention is further directed to an oligomer of the formulaCA-(AA)_(n)— wherein CA is the residue of an α-hydroxy carboxylic acid,each AA is the residue of an α-amino acid or derivative thereof whereingreater than one-half of the AA residues are derived from the groupconsisting of α-amino acids or derivatives thereof having the samechiral configuration, and n is at least 2.

The present invention is also directed to a process for providing ananimal with a food ration. The process comprises providing an oligomeror a co-oligomer prepared from a mixture containing an enzyme, anα-amino acid, and optionally, an α-hydroxy carboxylic acid or derivativethereof. The feed ration is administered to the animal by oraladministration, eye spray, placement in ear, placement in nasal cavity,and bucchal administration, sublingual administration, rectaladministration or injection.

The present invention is further directed to an orally administereddietary supplement comprising a vitamin, mineral, or nutrient that iscoated with an oligomeric coating. The coating comprises a residue of anα-hydroxy carboxylic acid bonded to a peptide by an amide linkage. Thepeptide comprises two or more independent α-amino acids independentlyselected from the group consisting of α-amino acids.

The present invention is further directed to a process for providing ananimal with a dietary supplement comprising a vitamin, mineral, ornutrient. The process comprises coating the vitamin, mineral or nutrientwith a composition to form a dietary supplement and administering thedietary supplement to the animal. The composition comprises a residue ofan α-hydroxy carboxylic acid bonded to a peptide by an amide linkage andthe peptide comprises two or more independent α-amino acidsindependently selected from the group consisting of α-amino acids.

The present invention is further directed to a process for purifying anenantiomeric mixture of α-amino acid or derivative thereof. The processcomprises forming a reaction mixture comprising (i) an enzyme, (ii) anenantiomeric mixture of α-amino acid or a derivative thereof, and (iii)an α-hydroxy carboxylic acid or a derivative thereof. A peptide reactionproduct is formed from the reaction mixture comprising (i) an oligomeror co-oligomer from the combination which incorporates one of themembers of the enantiomeric mixture of α-amino acid or derivativethereof in preference to a second enantiomer of the enantiomericmixture, and (ii) unreacted second enantiomer. The oligomer orco-oligomer and unreacted second enantiomer are then separated from thereaction product and each other.

The present invention is also directed to a process for purifying anα-hydroxy carboxylic acid enantiomer or derivative thereof in anenantiomeric mixture. The process comprises forming a reaction mixturecomprising (i)an enzyme, (ii) an enantiomeric mixture of an α-hydroxycarboxylic acid and (iii) an α-amino acid or a derivative thereof. Areaction product is formed from the reaction mixture comprising (i) aco-oligomer which preferentially incorporates a first enantiomer over asecond enantiomer of the enantiomeric mixture, and (ii)unreacted secondenantiomer. The co-oligomer and unreacted second enantiomer are thenseparated from the reaction product and each other.

Other objects and features of this invention will be in part apparentand in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a MALDI-TOF graph of methionine oligomers and co-oligomersfrom a papain catalyzed synthesis.

FIG. 2 is a MALDI-TOF graph of HMB-methionine co-oligomers from a papaincatalyzed synthesis.

FIG. 3 is a HPLC graph of methionine sulfone oligomers.

FIG. 4 is a HPLC graph of HMB-methionine sulfone co-oligomers after aincubation period of 10 minutes.

FIG. 5 is a HPLC graph of HMB-methionine sulfone co-oligomers after aincubation period of 24 hours.

FIG. 6 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of lysine oligomers synthesized in a reverse micellarsystem.

FIG. 7 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of HMB-lysine co-oligomers synthesized in a reversemicellar system.

FIG. 8 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of HMB-lysine co-oligomers synthesized in a 2-phasesystem.

FIG. 9 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of lysine oligomers synthesized in a 2-phase system.

FIG. 10 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of lysine oligomers synthesized in a 3-phase system.

FIG. 11 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of HMB-lysine co-oligomers synthesized in a 3-phasesystem.

FIG. 12 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of lysine oligomers synthesized in a reduced volume2-phase system.

FIG. 13 is a ion-pair liquid chromatography and MALDI-TOF massspectrometry graph of HMB-lysine co-oligomers synthesized in a reducedvolume 2-phase system.

FIG. 14A is a chromatogram of persulfonated methionine oligomers using aUV absorption detector.

FIG. 14B is a positive ion total ion chromatogram of persulfonatedmethionine oligomers.

FIG. 15A is a chromatogram of persulfonated HMB-methionine co-oligomersusing a UV absorption detector.

FIG. 15B is a positive ion total ion chromatogram of persulfonatedHMB-methionine co-oligomers.

FIG. 16 is a positive ion ESI spectra of (Met)₃ sulfone peak eluting at5.27 minutes.

FIG. 17 is a positive ion ESI spectra of (Met)₄ sulfone peak eluting at7.70 minutes.

FIG. 18 is a positive ion ESI spectra of (Met)₅ sulfone peak eluting at9.47 minutes.

FIG. 19 is a positive ion ESI spectra of (Met)₆ sulfone peak eluting at11.09 minutes.

FIG. 20A is a positive ion ESI spectra of (Met)₇ sulfone peak eluting at12.7 minutes.

FIG. 20B is a positive ion ESI spectra of (Met)₈ sulfone peak eluting at14.26 minutes.

FIG. 20C is a positive ion ESI spectra of (Met)₉ sulfone peak eluting at15.60 minutes.

FIG. 21A is a chromatogram of persulfonated methionine oligomers using aUV absorption detector.

FIG. 21B is a total ion chromatogram ESI-negative ion of persulfonatedmethionine oligomers.

FIG. 22A is a chromatogram of persulfonated HMB-methionine co-oligomersusing a UV absorption detector.

FIG. 22B is a total ion chromatogram ESI-negative ion of persulfonatedHMB-methionine co-oligomers.

FIG. 23 is a negative ion ESI spectra of HMB-(Met)₅ sulfone peak elutingat 11.57 minutes.

FIG. 24 is a negative ion ESI spectra of HMB-(Met)₆ sulfone peak elutingat 13.86 minutes.

FIG. 25 is a negative ion ESI spectra of HMB-(Met)₇ sulfone peak elutingat 15.31 minutes.

FIG. 26 is a bar graph of the relative distribution of (Met)_(n) whereinn is the number of methionine residues in the methionine oligomers.

FIG. 27 is a bar graph of the relative distribution of HMB-(Met)_(n)wherein n is the number of methionine residues in the HMB-methionineco-oligomers.

FIG. 28A is a positive ion ESI-MS spectra of HMB-methionine co-oligomerssynthesized with HMB methyl ester and methionine ethyl ester.

FIG. 28B is a negative ion ESI-MS spectra HMB-methionine co-oligomerssynthesized with HMB methyl ester and methionine ethyl ester.

FIG. 29 is a parent ion SSI-MS spectra HMB-methionine co-oligomerssynthesized with HMB methyl ester and methionine ethyl ester.

FIG. 30 is a daughter ion spectrum of (Met)₆-ethyl ester.

FIG. 31A is a positive ion ESI-MS spectra of tyrosine (Tyr)n oligomerswherein n is the number of tyrosine residues in the oligomers.

FIG. 31B is a negative ion ESI-MS spectra of tyrosine (Tyr)n oligomerswherein n is the number of tyrosine residues in the oligomers.

FIG. 32A is a positive ion spectra of HMB-tyrosine co-oligomers.

FIG. 32B is a negative ion spectra of HMB-tyrosine co-oligomers.

FIG. 33A is a positive ion ESI-MS spectra of leucine oligomers.

FIG. 33B is a negative ion ESI-MS spectra of leucine oligomers.

FIG. 34A is a positive ion ESI-MS spectra of HMB-leucine co-oligomers.

FIG. 34B is a negative ion ESI-MS spectra of HMB-leucine co-oligomers.

FIG. 35A is a positive ion ESI-MS spectra of HMB-phenylanalineco-oligomers.

FIG. 35B is a negative ion ESI-MS spectra of HMB-phenylanalineco-oligomers.

FIG. 36 is a graph of the effect of Aqueous : Non-Aqueous ratios on(Lys)_(n) oligomer yield wherein n is the number of lysine residues inthe oligomers in a two-phase system.

FIG. 37 is a bar graph of the effect of volumetric ratios on the degreeof (Lys)_(n) oligomer yield in a two-phase reaction system wherein n isthe number of lysine residues in the oligomers.

FIG. 38 is a graph of the effect of additive concentrations on (Lys)_(n)oligomer yield wherein n is the number of lysine residues in theoligomers.

FIG. 39 is a bar graph of the effect of additive concentrations on thedegree of (Lys)_(n) oligomerization wherein n is the number of lysineresidues in the oligomers.

FIG. 40 is a graph of the effect of substrate concentrations on(Lys)_(n) oligomer yield wherein n is the number of lysine residues inthe oligomers.

FIG. 41 is a bar graph of the distribution of lysine oligomers formed inreaction mixtures with varied substrate concentrations.

FIG. 42 is a graph of the effect of incubation time on total lysineoligomer yield.

FIG. 43 is a bar graph of the distribution of lysine oligomers formedafter different incubation time periods.

FIG. 44 is a graph of the effect of aqueous to non-aqueous solvent phaseratios on total lysine oligomer yield in a three-phase system.

FIG. 45 is a bar graph of the distribution of lysine oligomers formed inreaction mixtures at various aqueous to non-aqueous solvent ratios.

FIG. 46 is a graph of the effect of additive concentrations on the totallysine oligomers yield.

FIG. 47 is a bar graph of the distribution of lysine oligomers formedwith varied additive concentrations in a 2-phase system.

FIG. 48 is a graph of the total lysine oligomer yield formed afterdifferent incubation time periods in a 3-phase system.

FIG. 49 is a bar graph of the distribution of lysine oligomers formedafter a one hour incubation period in a 3-phase system.

FIG. 50 is a bar graph of the distribution of lysine oligomers formedafter a 24 hour incubation period in a 3-phase system.

FIG. 51 is a chromatogram of an enantiomeric mixture of methionine ethylester using a UV absorption diode array detector (DAD).

FIG. 52 is a chromatogram of a enantiomeric mixture of methionine ethylester and HMB-ethyl ester using a UV absorption diode array detector(DAD).

FIG. 53 is a chromatogram of an oligomer and co-oligomer hydrolyzateillustrating the presence of only the L-methionine enantiomer using a UVabsorption diode array detector (DAD).

FIG. 54 is a chromatogram of an oligomer and co-oligomer hydrolyzateillustrating the presence of only the L-HMB enantiomer using a UVabsorption diode array detector (DAD).

FIG. 55A is a positive ion ESI-MS spectra of the lactic acid-methionineoligomers prepared in Example 15.

FIG. 55B is a negative ion ESI-MS spectra of the lactic acid-methionineoligomers prepared in Example 15.

FIG. 56A is a positive ion ESI-MS spectra of the lactic acid-tyrosineoligomers prepared in Example 15.

FIG. 56B is a negative ion ESI-MS spectra of the lactic acid-tyrosineoligomers prepared in Example 15.

FIG. 57A is a positive ion ESI-MS spectra of the lactic acid-leucineoligomers prepared in Example 15.

FIG. 57B is a negative ion ESI-MS spectra of the lactic acid-leucineoligomers prepared in Example 15.

FIG. 58A is a positive ion ESI-MS spectra of the lactic acid-tryptophanoligomers prepared in Example 15.

FIG. 58B is a negative ion ESI-MS spectra of the lactic acid-tryptophanoligomers prepared in Example 15.

FIG. 59A is a positive ion ESI-MS spectra of the lacticacid-phenylalanine oligomers prepared in Example 15.

FIG. 59B is a negative ion ESI-MS spectra of the lacticacid-phenylalanine oligomers prepared in Example 15.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, it has been discovered thatoligomers and co-oligomers of α-hydroxy carboxylic acids and α-aminoacids may be prepared in an enzymatically catalyzed reaction.

The α-hydroxy carboxylic acid/α-amino acid oligomers enzymaticallysynthesized by the process of the present invention may possess alteredproperties from those of the α-amino acid monomers. For example, it hasbeen suggested that the α-hydroxy carboxylic acid/α-amino acidoligomers, unlike proteins, peptides, or amino acid monomers, are notrecognized in the rumen by ruminal microorganisms. As a result, theruminal microorganisms do not break down the oligomers and the oligomersare available for absorption by the ruminant.

Further, the solubility properties of many of the α-hydroxy carboxylicacid/α-amino acid oligomers of the present invention are also differentfrom their monomeric counterpart. Thus, while many α-amino acidmonomers, such as methionine, are soluble in water, the α-hydroxycarboxylic acid/α-amino acid oligomers and α-amino acid oligomers formedfrom methionine monomers are insoluble. This alteration advantageouslypermits the oligomers to be introduced in aqueous environments withoutbeing dissolved in the solution.

In general, the oligomers of the present invention comprise the residueof an α-hydroxy carboxylic acid bonded to the residue of an α-amino acidby an amide or an ester linkage. Thus, the oligomers correspond to thegeneral formula CA-(AA)_(n) wherein CA comprises the residue of anα-hydroxy carboxylic acid, (AA)_(n) is an oligomeric segment comprisingthe residue of one or more independent α-amino acids, n is at least 1and CA is bonded to (AA)_(n) by an amide or an ester linkage. Inaccordance with a preferred embodiment, the α-hydroxy carboxylic acid isbonded to the residue of an α-amino acid with an amide bond toeffectively create an α-amino acid oligomer that is “end-capped” by anα-hydroxy carboxylic acid residue.

If the reaction mixture does not contain an α-hydroxy carboxylic acid,an α-amino acid oligomer is formed that corresponds to the formula(AA)_(n) wherein each AA is the residue of one or more independentα-amino acids, n is at least 2 and the amino acid residues are bonded toeach other by an amide linkage or an ester.

It is important to note that when n is greater than 1, (AA)_(n) maycomprise more than one independent α-amino acid residue. Stated anotherway, (AA)_(n) comprises a peptide comprising two or more independentα-amino acids. Thus, the composition of the oligomer may beadvantageously tailored for specific applications. For example, in apreferred embodiment, the oligomer may be designed to meet the essentialamino acid requirements of a particular animal by incorporating two ormore different amino acid residues (e.g., methionine and lysineresidues) into an oligomer. Such an example is an oligomer comprisinglysine and methionine residues in a 3:1 ratio, which would meet theessential amino acid requirements of a ruminant.

Several variables affect the value of n, such as the amino acid monomerutilized, the reaction solution composition, and the method of isolatingthe co-oligomer products. Typically, n is less than 20. In someembodiments, n ranges from about 1 to about 10, more typically fromabout 2 to about 8 and, in some embodiments, from about 3 to about 5.For example, in oligomers comprising methionine residues, n typicallyranges from about 4 to about 12, with an average of about 6 to about 8.

The oligomers of the present invention may be obtained (and used) as adimer, trimer, tetramer, pentamer, hexamer, septamer, octamer, nonamer,decamer, etc. in which a residue of the α-hydroxy carboxylic acid islinked to a residue of an α-amino acid via an amide or ester linkage.Alternatively, an oligomeric segment may be obtained which is chemicallyor enzymatically linked to another moiety, for example, through theα-hydroxy group of the α-hydroxy carboxylic acid residue, the carboxyterminus of the α-amino acid residue (for oligomers comprising an amidelinkage between the α-hydroxy carboxylic acid residue and the α-aminoacid residue) or the amino terminus of the α-amino acid residue (foroligomers comprising an ester linkage between the α-hydroxy carboxylicacid residue and the α-amino acid residue).

In a preferred embodiment, the oligomer or oligomeric segmentcorresponds to the structure:

wherein

-   -   R¹ is hydrogen, hydrocarbyl or substituted hydrocarbyl,    -   R² is hydrogen, hydrocarbyl, substituted hydrocarbyl, or a        hydroxy protecting group,    -   R³ is hydrogen, hydrocarbyl or substituted hydrocarbyl,    -   each AA is the residue of an α-amino acid selected from the        group consisting of α-amino acids independently of any other        α-amino acid residue, and    -   n is at least 1.        α-hydroxy Carboxylic Acid Residue

In general, the oligomer or oligomeric segments of the present inventionmay comprise the residue of any α-hydroxy carboxylic acid. Preferredα-hydroxy carboxylic acids correspond to the general structureR¹R³C(OR²)COOH wherein R¹ is hydrogen, hydrocarbyl or substitutedhydrocarbyl; R² is hydrogen, a hydroxy protecting group, hydrocarbyl, orsubstituted hydrocarbyl; and R³ is hydrogen, hydrocarbyl or substitutedhydrocarbyl, preferably hydrogen. For example, the α-hydroxy carboxylicresidue may be the residue of any of the following naturally occurringα-hydroxy carboxylic acids (with R¹ for such acid being given inbrackets): lactic acid [—CH₃], mandelic acid [—C₆H₅], malic acid[—CH₂COOH], and tartaric acid [—CH(OH)COOH]. In addition, the α-hydroxycarboxylic acid residue may be the residue of an α-hydroxy acid analogof a naturally occurring α-amino acid, more preferably the residue ofthe α-hydroxy analog of an essential α-amino acid, and still morepreferably the residue of the α-hydroxy analog of methionine, i.e.,2-hydroxy-4-(methylthio)butyric acid.

In general, the α-hydroxy carboxylic acid residue may comprise theresidue of an α-hydroxy carboxylic acid having the D- configuration, theL- configuration, or a racemic or other mixture of the D- and L-isomers.In some embodiments, however, it is generally preferred that theα-hydroxy carboxylic acid residue comprise the residue of an α-hydroxycarboxylic acid having the L-configuration.

Further, it is important to note that the α-hydroxy carboxylic acidresidue incorporated into the oligomer may comprise the residue of morethan one α-hydroxy carboxylic acid. Thus, the residue may comprise ahomo-oligomer containing one or more α-hydroxy carboxylic acid monomersor a hetero-oligomer containing two or more independent α-hydroxycarboxylic acid monomers.

α-amino Acid Residue(s)

In general, the oligomers of the present invention may comprise theresidue of any α-amino acid. Preferred α-amino acids correspond to thegeneral structure R^(a)R^(b)C(NH₂)COOH wherein R^(a) is hydrogen,hydrocarbyl, substituted hydrocarbyl or heterocyclo; and R^(b) ishydrogen. For example, the α-hydroxy amino residue(s) may be theresidue(s) of any of the naturally occurring α-amino acids, e.g.,asparagine, glycine, alanine, valine, leucine, isoleucine,phenylalanine, proline, serine, threonine, cysteine, methionine,tryptophan, tyrosine, glutamine, aspartic acid, glutamic acid, lysine,arginine, and histidine. Preferably, the α-amino acid residue(s) includethe residue(s) of one or more essential α-amino acids, i.e., isoleucine,phenylalanine, leucine, lysine, methionine, threonine, tryptophan,histidine and valine. Still more preferably, the α-amino acid residue(s)include the residue(s) of methionine and/or lysine.

In general, the α-amino acid residue may comprise the residue of anα-amino acid having the D- configuration, the L- configuration, or aracemic or other mixture of the D- and L-isomers. In some embodiments,however, it is generally preferred that the α-amino acid residuecomprise the residue of an α-amino acid having the L-configuration.

Further, it is important to note that the α-amino acid residueincorporated into the oligomer may comprise the residue of more than oneα-amino acid. Thus, the residue may comprise a homo-oligomer containingone or more α-amino acid monomers or a hetero-oligomer containing two ormore independent α-amino acid monomers.

Enzymatic Oligomerization

The oligomers of the present invention are enzymatically synthesized ina mixture. The mixture comprises at least one α-hydroxy carboxylic acidor a derivative thereof, at least one α-amino acid or a derivativethereof, and an enzyme.

The α-hydroxy carboxylic acid may be present in the mixture as a freeacid or as a carboxylic acid derivative, e.g., the corresponding ester,acid halide, amide, anhydride, or ketene. Preferably, the α-hydroxycarboxylic acid and its derivatives have the formula R¹R³C(OR²)COY orR¹C(OR²)═C═O wherein R¹, R² and R³ are as previously defined and Y ishydroxy (for the free acid), halogen (for acid halide derivatives),hydrocarbyloxy (for ester derivatives), amino (for amide derivatives),and hydrocarbylcarboxy (for anhydride derivatives). In some embodiments,the α-hydroxy carboxylic acid is preferably present in the mixture inthe form of an ester, i.e., where Y is —OR⁵ and R⁵ is hydrocarbyl, morepreferably alkyl, alkene, or aryl, still more preferably lower alkyl. Inother embodiments, the α-hydroxy carboxylic acid is preferably presentin the mixture in the form of an amide, i.e., where Y is —NR⁶R⁷ and R⁶and R⁷ are independently hydrogen or hydrocarbyl, more preferably loweralkyl, still more preferably hydrogen.

The mixture may contain more than one α-hydroxy carboxylic acid species.Thus, for example, the mixture may contain the hydroxy analog ofmethionine (in one or more of its free acid, acid halide, amide,anhydride or ketene forms) and, in addition, one or more other α-hydroxycarboxylic acids such as lactic acid, mandelic acid, malic acid, ortartaric acid (in one or more of their respective free acid, acidhalide, amide, anhydride or ketene forms).

In addition to, or instead of α-hydroxy carboxylic acid monomers, themixture may further contain oligomers (e.g., dimers, trimers, tetramers,pentamer, hexamer, septamer, octamer, nonamer, decamer, etc.) of one ormore α-hydroxy carboxylic acids. For example, the mixture may contain ahomo-oligomer formed from HMB or another α-hydroxy carboxylic acid or ahetero-oligomer of an α-hydroxy carboxylic acid (e.g., HMB) and at leastone other α-hydroxy carboxylic acid.

Similarly, the α-amino acids may be present in the mixture as a freeacid or as a carboxylic acid derivative, e.g., the corresponding ester,acid halide, amide, anhydride, or ketene. In general, the α-amino acidand its derivatives have the formula R^(a)R^(b)C(NH₂)COY orR^(a)C(OR²)═C═O wherein R^(a), R² and R^(b) are as previously definedand Y is hydroxy (for the free acid), halogen (for acid halidederivatives), hydrocarbyloxy (for ester derivatives), amino (for amidederivatives), and hydrocarbylcarboxy (for anhydride derivatives). Insome embodiments, the α-amino acid is preferably present in the mixturein the form of an ester, i.e., where Y is —OR⁵ and R⁵ is hydrocarbyl,more preferably alkyl or aryl, still more preferably lower alkyl. Inother embodiments, the α-amino acid is preferably present in the mixturein the form of an amide, i.e., where Y is —NR⁶R⁷ and R⁶ and R⁷ areindependently hydrogen or hydrocarbyl, more preferably lower alkyl,still more preferably hydrogen.

The mixture may contain more than one α-amino acid species. Thus, forexample, the mixture may contain one α-amino acid (in one or more of itsfree acid, acid halide, amide, anhydride or ketene forms) and, inaddition, one or more other α-amino acids (in one or more of theirrespective free acid, acid halide, amide, anhydride or ketene forms). Byway of further example, the mixture may contain methionine (in one ormore of its free acid, acid halide, amide, anhydride or ketene forms)and, in addition, one or more other nutritionally important α-aminoacid(s) such as lysine, tryptophan and/or phenylalanine (in one or moreof their respective free acid, acid halide, amide, anhydride or keteneforms).

In addition to, or instead of α-amino acid monomers, the mixture maycontain oligomers (e.g., dimers, trimers, tetramers, pentamer, hexamer,septamer, octamer, nonamer, decamer, etc.) of one or more α-amino acids.For example, the mixture may contain a homo-oligomer formed frommethionine, lysine or other α-amino acid or a hetero-oligomer of anα-amino acid (e.g., methionine) and at least one other nutritionallyimportant α-amino acid such as lysine, tryptophan and/or phenylalanine.

The reaction mixture further comprises an enzyme. The enzyme may bedissolved in the mixture or, alternatively, it may be adsorbed orotherwise immobilized onto a variety of substrates. For example, theenzyme may be immobilized onto controlled pore glass, agarose,sepharose, nylon, or polyethylene glycol. Enzymes may also be adsorbed,for example, onto activated charcoal, ion exchange resins, silica,polyacrylamide, collagen, starch, bentonite, ultramembrane filters,cellulose, alumina, titania, and polyvinylchloride. In addition, enzymesmay be retained by entrapment, microencapsulation, liposome formation,hollow fiber, inorganic bridge formation, and aggregation.

The type of enzyme selected will determine the direction that anoligomerization process proceeds. For example, enzymes generallycharacterized as a protease when included in a reaction mixture alongwith, for example, an α-hydroxy carboxylic acid ethyl ester and anα-amino acid ethyl ester, will cause a peptide reaction product to beformed from the reaction mixture. The peptide reaction product comprisesan oligomer comprising α-amino acids and the α-hydroxy carboxylic acidbonded together by amide bonds. Examples of suitable protease enzymesinclude serine proteinases (e.g., Trypsin, α-Chymotrypsin, Elastase,Carboxypeptidase, and Subtilisin), thiol proteinases (e.g., Papain,Ficin, Bromelain, Streptococcal proteinase, Cathepsins, Calpains,Clostripain, and Actinidin), metalloproteinases (e.g., Thermolysin),acid proteinases (e.g., Pepsin, Penicillopepsin, Chymosin, Cathepsin,and Renin), liver esterase (e.g., pig liver esterase), alkalineprotease, carbonic anhydrase, nonribosomal peptide synthetase, thrombin,cardosins A or B, or pronase.

If, however, an enzyme such as a lipase enzyme is used, the reactionmixture containing the lipase enzyme, an α-hydroxy carboxylic acid, andan α-amino acid or derivative thereof, instead forms a polyesterreaction product. Enantioselective lipase enzymes may be obtained from avariety of microorganisms such as Candida cylindracea, Candidalipolytica, Candida antarctica (bacteria) and fungi such as Rhizopusoryzae, Aspergillus niger, and the like. The reaction product willtherefore comprise an oligomer wherein the α-amino acids and theα-hydroxy carboxylic acid are bonded together by ester bonds. If thereaction mixture comprises a lipase enzyme and an ester of an α-hydroxycarboxylic acid or a derivative thereof, an oligomer of α-hydroxycarboxylic acid will form wherein the monomers are linked together byester bonds.

In a preferred embodiment, the mixture contains an enzyme whichcatalyzes the formation of peptide bonds. Exemplary enzymes includeserine proteinases (e.g., Trypsin, α-Chymotrypsin, Elastase,Carboxypeptidase, and Subtilisin), thiol proteinases (e.g., Papain,Ficin, Bromelain, Streptococcal proteinase, Cathepsins, Calpains,Clostripain, and Actinidin), metalloproteinases (e.g., Thermolysin),acid proteinases (e.g., Pepsin, Penicillopepsin, Chymosin, Cathepsin,and Renin), liver esterase (e.g., pig liver esterase), alkalineprotease, carbonic anhydrase, nonribosomal peptide synthetase, thrombin,cardosins A or B, or pronase.

Enantioselective Enzymatic Oligomerization

It has further been found that the present invention may be utilized toenzymatically synthesize oligomers, co-oligomers, or segments thereof,consisting of α-hydroxy carboxylic acid isomers and α-amino acid isomersor α-amino acid isomers wherein one enantiomer of the α-hydroxycarboxylic acids, α-amino acids, or derivatives thereof is incorporatedinto the co-oligomer or oligomer in preference to the other enantiomer.Stated another way, it has been found that by using an enantioselectiveenzyme in the process of the present invention, peptide or esterreaction products comprising co-oligomers, oligomers or segments thereofcan be formed from a reaction mixture comprising an enantiomeric mixtureof α-hydroxy carboxylic acids, α-amino acids, or derivatives thereof,wherein one enantiomer of the enantiomeric mixture is incorporated intothe reaction product in preference to the other enantiomer of themixture.

As previously described for enzymatic oligomerization, the oligomers ofthe present invention are enantioselectively synthesized in a mixture.The mixture comprises at least one at least one α-amino acid or aderivative thereof as described above, an enantioselective enzyme; and,optionally a α-hydroxy carboxylic acid or a derivative thereof asdescribed above.

The α-hydroxy carboxylic acids and α-amino acids may be present in themixture as enantiomeric mixtures. An enantiomeric mixture containsenantiomeric pairs of the α-hydroxy carboxylic acids, α-amino acids, orderivatives thereof. The proportion of each species may vary from aracemic mixture that contains equal proportions of the D- and L-isomerconfigurations (e.g., 50% of the L-isomer and 50% of the D-isomer), toenantiomeric mixtures wherein one species is proportionally greater thanits opposite species (e.g., an enantiomeric mixture containing 70%L-isomer and 30% D-isomer).

In one embodiment of the present invention, the reaction mixturecontains a racemic mixture of α-amino acid. In another embodiment, thereaction mixture contains a racemic mixture of α-hydroxy carboxylicacid. In still another embodiment, the reaction mixture contains racemicmixtures of both α-hydroxy carboxylic acid and α-amino acid.

In general, the mixture contains an enzyme which enantioselectivelycatalyzes the formation of peptide bonds between α-amino acids havingidentical chiral configurations (e.g., L-isomers of amino acids). Thus,the co-oligomer or oligomer formed from the mixture comprises a residueof an α-hydroxy carboxylic acid bonded to a peptide by an amide linkageor an ester linkage, wherein the peptide comprises two or moreindependent α-amino acid residues having identical chiral configuration.

In another embodiment, the enzyme further enantioselectively catalyzesthe formation of the amide or ester linkage between the α-hydroxycarboxylic acid residue and the α-amino acid residue such that theoligomer comprises one α-hydroxy carboxylic acid enantiomer linked tothe α-amino acid oligomer in preference to another α-hydroxy carboxylicacid enantiomer. For example, a reaction mixture containing papain, anenantiomeric mixture of methionine ethyl ester isomers and anenantiomeric mixture of HMB ethyl ester isomers will form oligomersconsisting of L-HMB linked to one or more L-methionine residues. Theenantiospecificity of the enzyme is dependent upon the α-amino acid andα-hydroxy carboxylic acid being oligomerized. Exemplary enantioselectiveenzymes include thiol proteinases (e.g., Papain, Bromelain, Cathepsin s,Cathepsin b, and Cathepsin c) and serine proteinases (e.g., some formsof Subtilisin). In a preferred embodiment, the enantioselective enzymeenzymatically links the carboxy terminus of the α-hydroxy carboxylicacid to the amino terminus of the α-amino acid.

It is important to note that either of the α-hydroxy carboxylic acidresidue or the α-amino acid residues may comprise an oligomer (i.e., adimer, trimer, etc.) as described above and still be enantioselectivelyincorporated into the oligomers of the present invention. For example,if the enantioselective enzyme in the reaction mixture is suitable forincorporating oligomers comprising the L-enantiomer, the enzyme willcatalyze the oligomerization of any α-hydroxy carboxylic acid residue orα-amino acid residue having an L-configuration.

The enantioselective enzyme may be dissolved in the mixture or,alternatively, it may be adsorbed or otherwise immobilized onto avariety of substrates. For example, the enzyme may be immobilized ontocontrolled pore glass, agarose, sepharose, nylon, or polyethyleneglycol. Enantioselective enzymes may also be adsorbed, for example, ontoactivated charcoal, ion exchange resins, silica, polyacrylamide,collagen, starch, bentonite, ultramembrane filters, cellulose, alumina,titania, and polyvinylchloride. In addition, enzymes may be retained byentrapment, microencapsulation, liposome formation, hollow fiber,inorganic bridge formation, and aggregation.

In one embodiment, the oligomer formed from the enantioselectiveoligomerization comprises a composition comprising a residue of anα-hydroxy carboxylic acid bonded to a peptide by an amide or an esterlinkage, wherein the peptide comprises two or more α-amino acid residuesand each of the α-amino acids of the peptide are independently selectedfrom the group consisting of α-amino acids. Preferably, more than 50% ofthe α-amino acid residues in the peptide are of identical chirality and,more preferably, essentially all of the α-amino acid residues in thepeptide are of identical chirality.

In a preferred embodiment, the oligomer or oligomeric segment formed byan enantioselective enzyme corresponds to the structure:

wherein,

-   -   R¹ is hydrogen, hydrocarbyl or substituted hydrocarbyl,    -   R² is hydrogen, hydrocarbyl, substituted hydrocarbyl, or a        hydroxy protecting group,    -   R³ is hydrogen, hydrocarbyl or substituted hydrocarbyl,    -   each AA is the residue of an α-amino acid or derivative thereof        wherein greater than one-half of the AA residues are derived        from α-amino acids or derivatives thereof having the same chiral        configuration, and    -   n is at least 2.

For example, a mixture of papain, an enantiomeric mixture of HMB ethylester, and an enantiomeric mixture of D, L-methionine ethyl ester in areaction mixture has been found to form homo-oligomers of L-methionineand hetero-oligomers of L-HMB/L-methionine wherein the L-HMB is linkedthrough its carboxy terminus to the amino terminus of the methionineoligomer. The homo-oligomers and hetero-oligomers may be separated outof the solution by precipitation, filtration, selective extraction,column chromatography, lyophilization, and evaporation techniques.Typically, an oligomer comprising about nine methionine amino acids willprecipitate out of solution and may be easily filtered or centrifugedaway from the reacting mixture containing the free hydroxy acids andα-amino acids. Soluble methionine oligomers comprised of lower numbersof methionine residues can be separated from the free amino and hydroxyacids using membrane filtration. Once the reaction is allowed to run tonear completion, the remaining reaction mixture contains papain, asignificant amount of monomers of D-HMB ethyl ester and D-methionineethyl ester (e.g., about 95% of the total amount of HMB ethyl ester andmethionine ethyl ester isomers), and a small amount of L-HMB ethyl esterand L-methionine ethyl ester. Papain may be removed from the solution bysize exclusion chromatography or similar separation technique known inthe art. The remaining monomers of D-HMB ethyl ester and D-methionineethyl ester may be removed from solution by rotary evaporation forpurification or transformed to their respective L-isomer form throughbase racemization and recycled.

If the reaction mixture does not contain an α-hydroxy carboxylic acid,but does contain an enantioselective enzyme and an α-amino acid orderivative thereof, an α-amino acid oligomer is formed that correspondsto the formula (AA)_(n). AA is the residue of an α-amino acid comprisingtwo or more independent α-amino acids wherein greater than one-half ofthe AA residues are derived from α-amino acids or derivatives thereofhaving the same chiral configuration, n is at least 2, and the aminoacid residues are bonded to each other by an amide linkage. Typically, nwill be less than 20. In some embodiments, n will range from about 2 toabout 10, more typically from about 2 to about 8 and, in someembodiments, from about 3 to about 5. In methionine oligomers, forexample, n typically will range from about 4 to 12, with an average of 6to 8.

Enzymatic Reaction Mixtures

In one embodiment of the present invention, the enzymatic reaction iscarried out in a single phase, aqueous solution under conditionstypically employed in enzyme catalyzed reactions for the preparation ofoligomers and co-oligomers of α-amino acids. Such systems are typicallyused in enzymatic biochemical reaction. See, e.g., Lehninger, Nelson,and Cox, Principles of Biochemistry, 1993, Worth Publisher, NY, N.Y.

In a second embodiment of the present invention, the enzymatic reactionis carried out in a two-phase system comprising an aqueous phase and anorganic phase. In general, the organic phase comprises an organicsolvent selected from the group consisting of alkanes, alkenes, arylsand suitable derivatives thereof. See, e.g., Olmsted and Williams,Chemistry the Molecular Science, 1994, Mosby Publisher, St. Louis, Mo.

In a third embodiment of the present invention, the enzymatic reactionis carried out in a reverse micelle system. Such a system comprises acontinuous organic phase, a dispersed aqueous phase, and a surfactant toobtain and stabilize micelle phase. In general, the organic phasecomprises an organic solvent selected from the group consisting ofalkyl, aryl, and suitable derivatives thereof, and the surfactant isselected from the group consisting of ionic or non-ionic surfactants.Such reverse micelle systems are typically used for biotechnologicalreactions. See, e.g., Vicente, Aires-Barros, and Empis, J. Chem. Tech.Biotechnol. 1994, 60, 291.

In a fourth embodiment of the present invention, the enzymatic reactionis carried out in a three-phase system comprising an aqueous phase, afirst organic phase and a second organic phase with the two organicphases being immiscible. In general, the first organic phase comprisesan organic solvent selected from the group consisting of hydrocarbonsolvents and the second organic phase comprises an organic solventselected from the group consisting of halogenated hydrocarbon,perhalogenated hydrocarbon, and halogenated hydrocarbyl solvents. Suchthree phase systems are routinely used for chemical and biochemicalreactions.

In general, the reaction may be carried out over a relatively wide rangeof temperatures, e.g., about 4° C. to about 50° C., typically about 35to about 40° C. The pH of the aqueous phase is typically about 5.5 toabout 9. Depending upon whether the reaction is carried out in a singlephase, aqueous solution or in a multi-phase system, the ratio of thewater phase to the organic phase may range from 100:0 to 0.1:99.9 partsby weight, respectively. Reaction time may vary from minutes to hours(e.g., from about 10 minutes to about 24 hours or more) depending on thedesired yield and the synthesis may be achieved both with and withoutphysical agitation of the reaction mixture.

Separation

Specific oligomers and co-oligomers can be separated from the reactionmixtures through precipitation, filtration, selective extraction, columnchromatography, lyophilization, and evaporation techniques. Often, theoligomeric and co-oligomeric products are precipitates which may beeasily filtered or centrifuged away from the peptide and ester reactionproduct mixture containing free hydroxy acids and unreacted α-aminoacids. For example, soluble oligomer and co-oligomeric products can beseparated from reaction product mixture using membrane filtration.Alternatively, free amino acids and α-hydroxy acids may be removed fromthe product mixture using ion exchange or other applicablechromatographic technique. The selection of separation procedure isdependent on the desired oligomers and co-oligomers.

When enantioselective enzymes are utilized to form co-oligomersconsisting of α-hydroxy carboxylic acid isomers and α-amino acid isomershaving identical chiral configurations from a reaction mixturecontaining a racemic mixture of α-hydroxy carboxylic acid isomers and aracemic mixture of α-amino acids, the reaction mixture after thereaction is formed will contain the enzyme and a greater proportion ofthe non-selected enantiomers of α-hydroxy carboxylic acid and α-aminoacid than the non-selected enantiomers. The enzyme may be removed fromsolution by filtration and recycled, thereby leaving a solutionprimarily containing monomers of the non-selected enantiomers. Further,the non-selected enantiomers may be separated from the solution byrotary evaporation or by other methods known in the art. Afterseparation, the non-selected enantiomers may be transformed into themonomeric form of the selected isomer through base racemization andrecycled. For example, when the L-isomer of methionine is oligomerizedor co-oligomerized by papain in a reaction mixture containing a racemicmixture of methionine and HMB, at the end of the reaction, the mixturewill primarily be comprised of papain, D-methionine, and D-HMB. Papainmay be simply filtered from the reaction mixture by size exclusionchromatography leaving a solution primarily containing D-methionine(e.g., approximately 95% or more of the remaining racemic mixture ofmethionine) which may be isolated by rotary evaporation.

Alternatively, the process of the present invention can be used torecover the selected enantiomer from the separated oligomer. Forexample, the recovered oligomer may be hydrolyzed with acid to separatethe first, selected enantiomer or derivative thereof from otherhydrolyzates. The separated enantiomer may then be racemized andrecycled for further use.

Uses

Biological systems such as ruminants, poultry, swine, and aquaticanimals readily absorb and utilize the L-isomers of amino acids but areunable to utilize the corresponding D-isomer without first transformingthe D-isomer into the L-isomer through an oxidation followed bytransamination enzymatic reactions. As such reactions require additionaltime and energy to be expended by the animal before the amino acids canbe utilization by the animal, feed supplements of L-isomer oligomers andco-oligomers are advantageous as they can be utilized by the animal withminimal expenditure of energy, which ultimately improves the growth rateof the animals.

The enantioselective oligomerization and co-oligomerization of specificenantiomers of α-hydroxy carboxylic acids, α-amino acids, or derivativesthereof results in the purification of the species in the originalenantiomeric mixtures. First, by selectively oligomerizing orco-oligomerizing enantiomeric species, such as the L-enantiomers, theresulting oligomers and co-oligomers formed are therefore a more pureform of L-enantiomers. The L-enantiomers may then be isolated throughaddition of acid to the oligomers and co-oligomers thereby hydrolyzingthem into the L-enantiomers with which they are comprised. The L-isomersmay be further isolated from each other through chromatographic or otherseparation means known in the art.

Conversely, while the L-isomers are being selectively oligomerized andco-oligomerized, the reaction mixture contains an increasingly greaterproportion of the non-selected enantiomer species, for example, theD-enantiomers. Thus, as the reaction progresses, the remaining unreactedenantiomers, such as D- α-hydroxy carboxylic acids, D-α-amino acids, orderivatives thereof, may be recovered from the reaction mixture byrotary evaporation or other means. The D-isomers of α-hydroxy carboxylicacids, α-amino acids, or derivatives thereof may then be transformed totheir respective L-isomer form through base racemization and reused asreactants in additional oligomerization and co-oligomerizationreactions.

Depending upon the desired application, the α-hydroxy carboxylicacid/α-amino acid co-oligomer and α-amino acid oligomer compositions ofthe present invention may be provided to animals as an amino acidsupplement. The α-hydroxy carboxylic acid/α-amino acid co-oligomer andα-amino acid oligomer compositions may be fed or otherwise administeredorally, or sprayed into the eye, ear or nasal cavity of an animal,preferably a ruminant. Alternatively, the compositions may be injected,or administered bucchally (i.e., to the gums), sublingually (i.e.,beneath the tongue) or rectally.

The α-hydroxy carboxylic acid/α-amino acid co-oligomer compositions ofthe present invention or α-amino acid oligomer compositionsenzymatically formed in the absence of an α-hydroxy carboxylic acid mayalso be used to supplement the diets of animals not possessing rumens,such as poultry, swine, and aquatic animals. As with ruminants, thesecompositions may be fed or otherwise administered orally, bucchally,sublingually, rectally, sprayed into the eye, ear or nasal cavity of ananimal, or injected into the animal.

These compositions may be further used in aquaculture by applying thecompositions to an aquatic habitat in a particle size that is able to beingested by the target animal. The compositions may be used inaquaculture as particles of the α-amino acid oligomer or α-hydroxycarboxylic acid/α-amino acid co-oligomer itself or as an ingredient inthe animal's feed rations. While oligomers and co-oligomers may beutilized in varying sizes, feed mills typically manufacture feedsupplements in particle sizes of about 0.25 mm or more for incorporationinto feed rations. The feed ration pellets containing the oligomer orco-oligomer ingredient would be sized according to the animal to whichit is being fed. For example, fish, such as carp and trout, may be fedthe oligomer or co-oligomer compositions as an ingredient of a feedration that is applied to the surface of the water. Feed mills typicallyproduce fish feed rations in particle sizes that range between about 4mm to about 5 mm in diameter. Conversely, for smaller aquatic animals,such as shrimp, that cannot ingest large particles due to small mouthsize, the compositions may be applied to the surface of the water aspure forms of the oligomer or co-oligomer or as ingredients of a feedration in smaller feed particle sizes. Feed mills typically producerations for smaller aquatic animals in particles that are at least 1.6mm in diameter, preferably between about 2 mm to about 3 mm in diameter,more preferably between about 2.2 mm to about 2.4 mm in diameter. Thelength of the feed ration pellets are typically manufactured to be twoto three times the length of the diameter. While feed mills may producefeed rations in particular size ranges, the dimensions of the rationthat incorporates the oligomer or co-oligomer composition may be variedtherefrom without diminishing the effectiveness of the oligomer orco-oligomer.

In another embodiment, the α-hydroxy carboxylic acid/α-amino acidco-oligomer or α-amino acid oligomer compositions may be used as aprotective coating for vitamins, minerals, and other nutrientsupplements that are ingested by both humans and other animals, forexample, ruminants, poultry, swine, and aquatic animals. Vitamins andother nutritional supplements (e.g., vitamin A, acetate or palmitateester, and the like) which are ingested often must be protected againstacids and proteolytic enzymes present in the stomach and rumen in orderto be available for absorption by the animal in the intestine. Thesesupplements may often also be soluble in water or sensitive to oxidationsuch that they cannot remain in a solid form that can be ingested andutilized in an aqueous environment. Currently, protective coatings, suchas fat and gelatin based coatings, are applied to vitamins and nutrientsto protect against their degradation in the stomach or rumen ordissolution in water. These coatings may be made from animal productssuch as beef fat and gelatin. Such sources have recently come underscrutiny due to potential diseases carried by the animals which mayaffect the availability and quality of fats and gelatin used forcoatings.

The α-hydroxy carboxylic acid/α-amino acid co-oligomer or α-amino acidoligomer compositions provide a superior alternative to animal-basedcoatings. As some oligomer or co-oligomer compositions may be resistantto degradation in the stomach and rumen, as well as insoluble in water,they may be used as vitamins, minerals, and other nutrient coatings.Thus, in ruminants, the co-oligomer compositions may coat supplements inorder for the vitamins, minerals, and other nutrients to bypassmicrobial degradation that occurs in the rumen. Once in the smallintestine, the co-oligomer compositions are completely degraded whereinthe ruminant may absorb the α-hydroxy carboxylic acids, amino acids, andthe previously coated vitamins, minerals, and nutrients. Once absorbed,the ruminant may convert the α-hydroxy carboxylic acids from the coatingto its respective amino acid for utilization by the ruminant. Since theα-hydroxy carboxylic acid/α-amino acid co-oligomer and α-amino acidoligomer coatings are enzymatically synthesized, they do not introducethe risk of infecting the ruminant with a disease that may have beencarried by an animal from which a fat or gelatin based coating isderived.

For non-ruminants, gastric acids and enzymes present in the stomachbegin to degrade the coating as it passes through the stomach to theintestine. Once in the intestine, the coating is completely degraded,and the previously encapsulated vitamins, minerals, and other nutrientsmay be absorbed.

Some supplements, such as vitamin A, are soluble in water. Leftuncoated, the soluble supplements would dissolve into the surroundingwater and pollute it rather than provide the aquatic animals with thevitamins, minerals, and other nutrient supplements they need. As theα-hydroxy carboxylic acid/α-amino acid co-oligomer or α-amino acidoligomer compositions are also insoluble in water, they also may bebeneficially used to coat vitamins, minerals and other nutrientsupplements for use in aquaculture.

The application of α-hydroxy carboxylic acid/α-amino acid co-oligomer orα-amino acid oligomer coatings to vitamins and other nutritionalsupplements may be achieved by methods known in the art. For example,the oligomer or co-oligomer may be dissolved in a volatile solvent andsubsequently spray coated on a fluidized bed of the supplements. As thesolvent evaporates, a coating of α-hydroxy carboxylic acid/α-amino acidco-oligomer or α-amino acid oligomer remains on the supplements whichmay then be provided to the animal.

Definitions

The term “aquaculture” refers to the cultivation of aquatic animalsincluding, but not limited to, freshwater and salt water fish (e.g.,carp, trout, catfish, bass, sea bass, cod, salmon, and fish relatedthereto) and crustaceans (e.g., shrimp, crabs, lobster, freshwatershrimp, and the like).

The terms “hydrocarbon” and “hydrocarbyl” as used herein describeorganic compounds or radicals consisting exclusively of the elementscarbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Preferably, thesemoieties comprise 1 to 20 carbon atoms.

The “substituted hydrocarbyl” moieties described herein are hydrocarbylmoieties which are substituted with at least one atom other than carbon,including moieties in which a carbon chain atom is substituted with ahetero atom such as nitrogen, oxygen, silicon, phosphorous, boron,sulfur, or a halogen atom. These substituents include halogen,heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, protectedhydroxy, keto, acyl, acyloxy; nitro, amino, amido, nitro, cyano, andthiol.

The alkyl groups described herein are preferably lower alkyl containingfrom one to six carbon atoms in the principal chain and up to 20 carbonatoms. They may be straight or branched chain and include methyl, ethyl,propyl, isopropyl, butyl, hexyl and the like.

The alkenyl groups described herein are preferably lower alkenylcontaining from two to six carbon atoms in the principal chain and up to20 carbon atoms. They may be straight or branched chain and includeethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and thelike.

The alkynyl groups described herein are preferably lower alkynylcontaining from two to six carbon atoms in the principal chain and up to20 carbon atoms. They may be straight or branched chain and includeethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.

The terms “aryl” or “ar” as used herein alone or as part of anothergroup denote optionally substituted homocyclic aromatic groups,preferably monocyclic or bicyclic groups containing from 6 to 12 carbonsin the ring portion, such as phenyl, biphenyl, naphthyl, substitutedphenyl, substituted biphenyl or substituted naphthyl. Phenyl andsubstituted phenyl are the more preferred aryl.

The terms “halogen” or “halo” as used herein alone or as part of anothergroup refer to chlorine, bromine, fluorine, and iodine.

The terms “heterocyclo” or “heterocyclic” as used herein alone or aspart of another group denote optionally substituted, fully saturated orunsaturated, monocyclic or bicyclic, aromatic or nonaromatic hydrocarbongroups having at least one heteroatom in at least one ring, andpreferably 5 or 6 atoms in each ring. The heterocyclo group preferablyhas 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogenatoms in the ring, and may be bonded to the remainder of the moleculethrough a carbon or heteroatom. Exemplary heterocyclo include furyl,thienyl, pyridyl and the like. Exemplary substituents include one ormore of the following groups: hydrocarbyl, substituted hydrocarbyl,keto, hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy,alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, and thiol.

The acyl moieties described herein contain hydrocarbyl, substitutedhydrocarbyl or heterocyclo moieties.

The terms “hydroxyl protecting group” and “hydroxy protecting group” asused herein denote a group capable of protecting a free hydroxyl group(“protected hydroxyl”) which, subsequent to the reaction for whichprotection is employed, may be removed without disturbing the remainderof the molecule. A variety of protecting groups for the hydroxyl groupand the synthesis thereof may be found in “Protective Groups in OrganicSynthesis” by T. W. Greene, John Wiley and Sons, 1981, or Fieser &Fieser. Exemplary hydroxyl protecting groups include acetyl (Ac), benzyl(PhCH₂—), 1-ethoxyethyl (EE), methoxymethyl (MOM), (methoxyethoxy)methyl(MEM), (p-methoxyphenyl)methoxymethyl (MPM), tert-butyldimethylsilyl(TBS), tert-butyldiphenylsilyl (TBPS), tert-butoxycarbonyl (Boc),tetrahydropyranyl (THP), triphenylmethyl (Trityl, Tr),2-methoxy-2-methylpropyl, benzyloxycarbonyl (Cbz), trichloroacetyl(OCCCl₃), benzyloxymethyl (BOM), tert-butyl (t-Bu), triethylsilyl (TES),trimethylsilyl (TMS), and triisopropylsilyl (TIPS).

The abbreviation “HMB” shall mean the 2-hydroxy analog of methionine,i.e., 2-hydroxy-4-(methylthio)butyric acid.

The terms “chiral,” “chiral configuration,” and “enantiomer” refer to aparticular stereoisomer of a molecule. For example, L-methionine andL-HMB.

The term “identical chirality” or “identical chiral configuration”refers to the chiral carbon of two or more molecules having the samestereoisomeric configuration. For example, all L-isomers of α-amino acidhave identical chiral configuration. Thus, in the general α-amino acidstructure, R^(a)R^(b)C(NH₂)COOH, wherein R^(a) is hydrogen, hydrocarbyl,substituted hydrocarbyl or heterocyclo; and R^(b) is hydrogen, the—COOH, —NH2, R^(a), and R^(b) constituents of L-isomers of α-amino acidhave the same spatial arrangement around the chiral carbon. Similarly,the two or more L-enantiomers of a specific α-hydroxy carboxylic acid,such as two molecules of L-HMB, will have identical configuration toeach other.

The term “enantioselective” refers to the selection of a specificenantiomer of an enantiomeric mixture and interactions with saidenantiomer. For example, an enantioselective enzyme, such as papain,selectively catalyzes the linkage of L-methionine ethyl esters to forman oligomer of L-methionine residues.

EXAMPLES

The following Examples set forth one approach that may be used to carryout the process of the present invention. Accordingly, these examplesshould not be interpreted in a limiting sense.

Example 1 Synthesis of Methionine Oligomers and HMB-MethionineCo-Oligomers

This example demonstrates the enzymatic synthesis of oligomerscomprising methionine and co-oligomers comprising HMB-methionine, aswell as their characterization using reverse-phase HPLC and matrixassisted laser desorption ionization-time of flight mass spectroscopy(MALDI-TOF MS) analysis.

Co-Oligomerization

In a first synthesis, the experiment was generally conducted inaccordance with reaction conditions for the papain-catalyzedoligomerization of methionine analogs as described by Arai et al. inAgric. Biol. Chem., 43(5), 1069-1074 (1979). The oligomerizationcomprised forming a reaction mixture at a temperature of 37° C.consisting of nanopure filtered water (10 mL) containing HMB ethyl ester(0.7 M) and methionine ethyl ester (0.7 M) along with L-cysteine (0.1M), EDTA (10 mM), sodium citrate (1M) and 1% papain (by weight of themonomer) at a pH of 5.5. The mixture was allowed to incubate for 24hours. After 10 minutes, aliquots were removed at regular intervals tomonitor the degree of oligomerization and co-oligomerization and thedisappearance of the substrate.

In a second synthesis, the experiment was conducted in accordance withreaction conditions for the papain-catalyzed oligomerization ofmethionine analogs as described by Jost et al. in Helv. Chim. Acta, 63(1980) 375-384 (1980). The oligomerization comprised forming a reactionmixture consisting of L-methionine ethyl ester (5 g) and HMB ethyl ester(5 g) dissolved in nanopure water (50 mL) containing sodium bicarbonatebuffer (0.1 mole) and L-cysteine (4 mmole). The pH was adjusted to 9 andthe solution was made up to 100 mL and incubated for 24 hours at 37° C.after adding papain (2 g).

Analysis of Oligomers

In all cases, aliquots were removed from the oligomerization reactionmixtures and heated to 80° C. for 10 minutes to denature the enzyme. Themixture was centrifuged and the supernatant was analyzed on areverse-phase HPLC to monitor the synthesis of methionine oligomers oforder 3 or less along with the disappearance of the substrate. Attemptsat resolving the higher order oligomers with RPLC and gel permeationliquid chromatography (GPC) were unsatisfactory especially for oligomerswith 4-10 monomer residues. For example, the experiments revealed thatunderivatized oligomers could not be eluted from C-18 or C-8 columnswith the common mobile phases due to poor solubility of oligomers inthese mobile phases. The oligomers were soluble in dimethyl sulfoxide(DMSO) and tetrahydrofliran (THF) a common mobile phase in GPC forseparations. However, oligomers with less than ten residues could not beresolved from the solvent in GPC separations. A persulfonation procedurewas therefore adopted. Persulfonation of oligomers enhanced the polarityof the oligomers to a point that these could be separated on a C- 18column with a moderately polar mobile phase (M. Spindler, R. Stadler andH. J. Tanner, J. Agri. Food Chem., 32(6) (1984)1366-1371).

Persulfonation of Oligomers

The problem of analysis of higher order oligomers was addressed by theoxidation of the methionine and the HMB to their relatively hydrophilicsulfones with performic acid. The mixture was washed thoroughly until notraces of the monomers and the salts were left behind. The mixture wasthen freeze dried and a part of it was subjected to persulfonation witha method which was adapted from a procedure outlined by Spidler andcoworkers. The procedure involved oxidation of all sulfide moieties inthe oligomers with performic acid. The performic acid for the purposewas prepared by oxidation of formic acid (HCOOH) with hydrogen peroxide(H₂O₂). A solution of 30% H₂O₂ (0.5 mL) was mixed with 88% HCOOH (4.5mL) and phenol (25 mg). The mixture was allowed to stand for 30 minutesat room temperature. After 30 minutes, the mixture was cooled to 0° C.for 15 minutes in an ice bath. The finely divided oligomer powder (10mg) was then contacted with the performic acid mixture in the ice bath.After stirring for 15 minutes, the oligomer-performic acid mixture wasplaced in a refrigerator overnight. The excess performic acid wasreduced with 48% hydrobromic acid (0.7 mL). The residual bromine andformic acid were removed with a rotary evaporator at 50-60° C.

Liquid Chromatography

The oligomer sulfone residues in the rotary evaporator round bottomflasks were dissolved in a 40:60 acetonitrile/water mixture (5 mL) andfiltered through a membrane filter. A 10 μL aliquot of the solution wasinjected into a HPLC. The separation of persulfonated oligomers wasachieved with a C-18 column using a phosphate buffer-acetonitrile mobilephase. A linear gradient was used to facilitate separations. In thisgradient the mobile phase composition was changed from 100% eluant A(phosphate buffer, pH 6.5) to 60% A and 40% B (20% Acetonitrile) in 20minutes. The mobile phase flow rate was maintained at 1 mL min⁻¹. Theseparated oligomers were detected with a UV/VIS diode array detector.

TOF Experiments

Aliquots of purified oligomers dissolved in DMSO were introduced intothe mass spectrometer along with a thioglycerol matrix. The massspectrometer operating parameters were:

Accelerating Potential+20 KV

Grid Voltage 80%

Low Mass Gate 191.0

Flight tube pressure 3.3 e⁻⁷ torr

MALDI-TOF Analysis

The MALDI-TOF spectra of methionine (Met) oligomers are shown in FIG. 1.The spectra contain distinct ions which are separated by mass 131. Thismass (131) represents the repeating Met moiety (C₅H₉N O S), since themasses of the N and C terminal methionine residues are 132 and 148respectively. Therefore, a methionine hexamer (Met)⁶,(^(N)Met)-(Met)₄-Met^(C))+H⁺ should appear at m/z 805 and (Met)⁷ shouldappear at m/z 936. However, the m/z values of the dominant ions did notcorrespond to this series, instead, one set of dominant ions appeared atm/z 826, 957, 1088, 1219, 1350 and 1481. These ions most likelycorrespond to ((Met)^(n)+Na⁺), where n is an integer between 6 and 11.The second group of ions appeared at m/z 724, 855, 986, 1117, 1248 and1379. These ions most likely correspond to the series(^(N)Met-(Met)-Met-O-C₂H₅)+Na⁺. A third set of ions appeared at m/z 739,870, 1001 and 1134, these ions most likely correspond to^(N)Met-(Met)_(n)-Met-O-C₂H₅+K⁺. A fourth set of unidentified ions seemto be present at regular intervals in the clusters and which might beassigned the mass values, 842, 973, 1104, 1235 and 1366 corresponding tothe series ^(N)Met-(Met)_(n)-Met^(C))+K⁺. In all these cases “n” variedbetween 6 to 11.

The spectra of HMB-Met co-oligomers is shown in FIG. 2. In this spectra,ions corresponding to the series (N^(Met)-(Met)_(n)-Met^(C))+Na⁺,(^(N)Met-(Met)_(n)-Met-O-C₂H₅)+Na⁺, ^(N)Met-(Met)_(n)-Met^(C)+K⁺, and^(N)Met-(Met)_(t)C₂H₅+K⁺ were readily observed. However, the ions, whichshould correspond to (HMB-(Met)_(n)-Met^(C))+H⁺ or +Na⁺ m/z 806, 937 and1118; 827, 958 and 1089 were not observed in the spectra. The apparentabsence of these ions, however, does not necessarily mean the absence ofHMB-(Met)^(n) co-oligomers in the product mixture. The absence of theions can be attributed to two factors. The first relates to the lowresolving power of the MALDI-TOF MS, which would prevent the resolutionof the H³⁰ HMB-(Met)^(n)-Met^(C) ions at m/z 806, 937, 1118 from theH^(+ N)Met-(Met)^(n)-Met^(C) ions at m/z 805, 936, 1117. The second andmore probable cause is the low intensity of the H⁺HMB-(Met)^(n)-Met^(C)ion due to the absence of a good protonation site in HMB-(Met)^(n)co-oligomers.

HPLC Separations of Oligomers Sulfones

The chromatographic separations of poly-methionine sulfones are shown inFIG. 3. A number of well-resolved peaks can be readily observed. Ofthese, nine did not appear in the reagent blank and most likelyrepresent the poly-methionine sulfones. This chromatographic separationis nearly identical to the chromatographic separations reported by Kasaiet al. (T. Kasai, T. Tanaka, and S. Kiriyama, Biosci. Biotech. Biochem.,56(11) (1992) 1884-1885). However, because of a difference in theseparation column or variations in the eluant composition, the retentiontimes reported by Kasai et al. for most oligomers were approximately0.5-0.6 minutes longer than retention times observed by the presentinventors.

The chromatographic separation of HMB-poly-methionine sulfones is shownin FIG. 4. This chromatogram contained a number of peaks, which were notpresent in the poly methionine sulfone chromatogram. This indicates thatHMB is incorporated in the (Met)^(n) oligomer. The incorporation mostlikely occurs at the N-terminal end. The resulting HMB-(Met)^(n)co-oligomers, with the terminal hydroxyl, should be less polar than thecorresponding (Met)^(n) oligomers with the terminal amine moiety.Therefore, the HMB containing co-oligomers should elute later than thecorresponding Met oligomers and this appears to be the case. The elutiontimes for methionine sulfones and HMB methionine sulfones are given inTable 1. The chromatographic separations of methionine oligomer(sulfones) obtained after different incubation periods indicate that therelative abundance of methionine oligomers is dependent on theincubation period. The abundance of longer chain co-oligomers was higherin co-oligomers obtained after 24 hours incubation (FIG. 5) relative tothe co-oligomers obtained after 10 minutes incubation (FIG. 4). It canbe readily observed that the concentrations of longer chain co-oligomersincreased with an increase in the incubation period. Chromatographicresults also indicate that presence of HMB may affect the relativedistribution of methionine oligomers. These results are significant inlight of the reports in the literature, which suggest that the uptake ofmethionine oligomers is dependent on the size of the oligomers. TABLE 1Elution Times of Met Oligomer and HMB-Met Co-Oligomer Sulfones ElutionTime (mins) Elution Time (mins) Oligomer Present Study Kasai et al.(Met)₄ 10.0 NR HMB-(Met)₃ 11.8 NA (Met)₅ 13.4 14.0 HMB-(Met)₄ 15.1 NA(Met)₆ 16.8 17.8 HMB-(Met)₅ 18.5 NA (Met)₇ 20.1 21.0 HMB-(Met)₆ 21.8 NA(Met)₈ 23.3 24.0 HMB-(Met)₇ 24.9 NA (Met)₉ 26.5 26.9 HMB-(Met)₈ 28.6 NA(Met)₁₀ 31.1 29.5 HMB-(Met)₉ 34.2 NANR: Not ReportedNA: Not Available

Example 2 Oligomerization and Co-Oligomerization of Lysine and HMB

This example demonstrates four alternative procedures for the enzymaticsynthesis of oligomers comprising lysine and co-oligomers comprisingHMB-lysine. The experiment was designed to compare three novel synthesisprocedures to that of Puigserver et. al.¹ who reported a procedure forpapain catalyzed polymerization of lysine.

In general, protease-catalyzed synthesis of water insoluble amino acidoligomers in aqueous media is driven by precipitation. The synthesis ofwater soluble oligomers of amino acids, such as lysine can be controlledonly in mixed phase systems where the equilibria is shifted in favor ofthe synthesis of polypeptides due to enhanced partitioning of peptide inthe organic phase. Puigserver et. al. reported a procedure for papaincatalyzed polymerization of lysine which involved the binding of papainto modified PEG (MW 2000 or 5000). The bound enzyme was then used tosynthesize poly lysine in a two phase reaction mixture. The followingsummarizes the experiment to use Puigserver's method to explore thefeasibility of the co-oligomerization of lysine and HMB in comparison tothree novel synthesis methods.

PEG bound Papain system (Puigserver's Method): 10 mM of substrate wasadded to 98 mL of toluene along with 0.8 mL of Diisopropyl amino ethyland 0.2 mL of mercaptoethanol, followed by 17 mM of PEG₂₀₀₀ modifiedPapain. The mixture was allowed to incubate for 24 hours, before beingevaporated and redissolved in deionized water and analyzed on a ion-pairliquid chromatography column.

Two Phase Toluene: Water System: This solvent system was evaluated withvaried phase ratios, two of which are described below:

-   a) 10 mM of substrate was added to 98 mL of toluene along with 0.8    mL of Diisopropyl amino ethyl and 0.2 mL of mercaptoethanol,    followed by 1 mL of aqueous papain suspension. The mixture was    allowed to incubate for 24 hours, before being evaporated and    redissolved in DI water and analyzed on a ion-pair liquid    chromatography column.-   b) 100 mM of substrate was added to 8.9 mL of toluene along with    0.08 mL of Diisopropyl amino ethyl and 0.02 mL of mercaptoethanol.    This was followed by 1 mL of aqueous papain suspension, which    resulted in a two phase system. The mixture was allowed to incubate    for 24 hours, before being evaporated and redissolved in DI water    and analyzed on an ion-pair liquid chromatography column.    Reverse Micellar System: 10 mM of substrate was dissolved in 98 mL    of a reverse micellar solution containing 150 mM of AOT (3.33 g),    0.8 mL of diisopropyl amino ethyl and 0.2 mL of mercaptoethanol in    isooctane. 1 mL of aqueous papain solution was added to the mixture    and allowed to incubate for 24 hours, at the end of which the    mixture was heated to denature the enzyme and the oligomeric and    co-oligomeric products extracted with 1 M NaCl solution. The    solution was later analyzed on a ion-pair liquid chromatography    column.    Three Phase DFP:Octane:Water System: To a two phase system    comprising of 4.45 mL of DFP and 4.45 mL of octane was added 100 mM    of substrate along with 0.08 mL of Diisopropyl amino ethyl and 0.02    mL of mercaptoethanol. The addition of 1 mL of aqueous papain    suspension which is insoluble in either of the phases converts this    system to a three phase system. The mixture was allowed to incubate    for 24 hours, before being evaporated and redissolved in DI water    and analyzed on a ion-pair liquid chromatography column

Results: The yield and the degree of oligomerization andco-oligomerization were determined with ion-pair liquid chromatographyand MALDI-TOF mass spectrometry. These results appear in FIGS. 6 to 13.In general, Puigserver's method was found to be cumbersome and did notyield any discernable HMB-lysine co-oligomers. Reaction conditions andresults are further summarized in Table 2. TABLE 2 Procedure for thevarious methods used to synthesize lysine oligomers and HMB-lysineco-oligomers Lysine Oligomerization HMB-lysine Co-oligomerizationComponents Pgsr^(f) 2-f^(g) RM^(h) 3-f^(i) RV 2-f^(j) Pgsr^(f) 2-f^(g)RM^(h) 3-f^(i) RV 2-f^(j) LysEE•2HCl (mM) 10 10 10 100 100 5 10 10 50 50HMB analog mM 5 10 10 50 50 i-Pr₂NH₂Et (% v/v)^(a) 0.8 0.8 0.8 0.8 0.80.8 0.8 0.8 0.8 0.8 SCH₂CH₂OH (% v/v)^(b) 0.2 0.2 0.2 0.2 0.2 0.2 0.20.2 0.2 0.2 Papain (% v/v)^(c) 1 1 10 10 1 1 10 10 Toluene (% v/v) 98 9889 98 98 0 89 Isooctane (% v/v) 98 98 DFP (% v/v)^(d) 44.5 44.5 Octane(% v/v) 44.5 44.5 AOT (mM)^(e) 150 150 PEG-Papain (mM) 17 17 Yields (%)0 17 22 95 90 0 17 22 95 90^(a)Di-isopropyl amino ethyl^(b)Mercaptoethanol^(c)Aqueous suspension of papain obtained from Sigma^(d)Decafluoropentane^(e)Aerosol-OT, Dioctyl sulfor succinate^(f)Puigserver's method^(g)Two-phase method^(i)Three Phase Method^(h)Reverse Micellar method^(i)Three Phase method^(j)Reduced volume (10%) m¹ Anne Frejancic, Antoine Puigserver and Hubert Gaertner,Papain-Catalyzed Polymerization of Amino Acids in Low Water OrganicSolvents, Biotech. Lett., 1991, 13 (3), 161-166.

Example 3

HMB-methionine and HMB-lysine co-polymers were synthesized enzymaticallythrough a papain-catalyzed reaction along with poly-methionine andpoly-lysine (as controls) as described in Examples 1 and 2. Thebiological release of the amino acids from the oligomers was examinedusing several digestive enzymes including pepsin, trypsin, chymotrypsin,intestinal peptidase and carboxypeptidase. The oligomers were dissolvedat 10 mg/mL in 0.15 HCl (pH 2.5) or 50 mM KPO₄ (pH 7.5). Samples (0.5mL) were incubated with 10 units of each enzyme for 2 hours at 37° C.The extent of digestion was quantified by measurement of newly releasedamino groups and their reaction with o-Phthalaldehyde (OPA) and2,4,6-trinitrobenzene sulfonic acid (TNBSA). Acid hydrolysis wasprepared by complete hydrolysis of 10 mg/mL polymers in 6 M HCl for 24hours at 110° C. Results are summarized below in Table 3.

Results show that HMB-methionine and HMB-lysine can be hydrolyzed bystrong acid and heat. HMB-met is digested only 3.5% by pepsin and not atall by the other proteases. Poly-lysine can be digested by intestinalpeptidase (20% in 2 hours at 37° C.) but not by other proteases.HMB-lysine is not digested by any of the proteases tested. Inconclusion, these data suggest the lack of enzymatic digestion ofHMB-met and HMB-lysine co-oligomer was caused by a structural differenceinstead of solubility of the co-oligomers. TABLE 3 ENZYMATIC DIGESTIONOF AMINO ACID POLYMERS Poly-Lys Poly-Lys Enzyme (˜8mer) HMB-met Poly-metHMB-Lys (˜4mer) pepsin 0 0.030 (3%) 0.062 (15%) 0 0 trypsin 0.008 (15%)0 0 0 0 chymotrypsin 0 0 0 0 0 intestinal 0.013 (25%) 0 0.052 (13%) 0(TNBSA, ˜20%) peptidase carboxy- 0 0 0 0 0 peptidase A acid 0.052 0.8680.406 0.063 0.093 hydrolysis (−initial value)Readings were from OPA analysis of 20 μg samplesHydrolysis was done at 37° C. for 2 hours(%) refers to % of acid hydrolysis number

Example 4 Characterization of Methionine Oligomers and HMB-MethionineCo-Oligomers

Met oligomers and HMB-Met co-oligomers produced through papain mediatedenzymatic reactions at pH 5.5 and pH 9.0 according to the proceduredescribed in Example 1 were subjected to persulfonation. Persulfonederivatives were separated with the reverse phase liquid chromatography(RPLC). The separated oligomers and co-oligomers were monitored with aUV absorption spectrophotometeric detector and an electrosprayionization interface (ESI) mass spectrometer. The absorption wavelengthwas set at 210 nm. The mass spectrometer was operated in positive andnegative ion modes. The outputs of the UV absorption detector and thepositive ion ESI-MS are shown in FIGS. 14 and 15.

The chromatogram of (Met)n persulfones obtained with the UV detector andthe positive ion total ion chromatogram (TIC) were similar to thechromatograms obtained from earlier with a earlier experiments using aRPLC-Diode Array Detector (DAD) system. These results had indicated theformation of Met homo-oligopeptides and HMB-Met co-oligomers. Theresults were supported by data obtained from the matrix assisted laserdesorption ionization-mass spectrometry (MALDI-MS) experiments.

The experiments with ESI-MS in the positive ion mode confirmed theformation of methionine oligomers. The ESI-MS spectrum for individual LCpeaks provides conclusive evidence for the formation of (Met) to (Met)oligomers. The mass difference between in the molecular masses ofsuccessive oligomers was found to be 163, which corresponds to thesulfonated methionine residue.

Spectra corresponding to different peaks are shown in FIGS. 16-20. Thegeneral formula for the separated oligomers is:

The positive ion TIC of HMB-(Met)n co-oligomers obtained with ESI-MS didnot contain extra peaks observed in the LC-UV chromatogram. The spectraof individual peaks in the HMB-Met co-oligomers did not provide anyevidence for HMB-(Met)n co-oligomers formation. These results were notunexpected, the lack of pseudo-molecular positive ions in the MALDI-TOFspectra of HMB-(Met)n in the earlier experiments had led us to theconclusion that HMB is attached at the N-terminal end of thepolymethionine chain. The lack of protonated ions in the HMB-Metco-oligomers is the result the weak proton affinity of the terminalhydroxyl group.

The confirmation of the HMB-(Met)n was obtained by monitoring negativeions formed through electron attachment to the (Met)n and HMB-(Met)nchains. The TIC of HMB-(Met)n in this case contained extra components(peaks) which corresponded to the extra peaks observed in the LC-UVchromatograms FIGS. 21 and 22.

A few representative spectra for the HMB-(Met)n peaks are shown in FIGS.23-25. As expected, the molecular ions for HMB-(Met)n appear at one massunit higher than the corresponding (Met)n ions. In addition, theretention times of HMB-(Met)n peaks are longer than the corresponding(Met)n peaks. This is to be expected since the terminal amine group ofthe (Met)n imparts higher polarity to methionine oligomers than theterminal hydroxyl to the HMB-(Met)n co-oligomers.

The presence of sulfonated methionine residue in both (Met)n oligomersand the HMB-(Met)n co-oligomers chains is again revealed by massdifference of 163 amu between the molecular masses of the separatedchromatographic peaks. The mass difference corresponds to the mass ofthe methionine sulfone residue.

Both the positive ion and negative ion LC-ESIMS data show that thepredominant (Met)n are composed of four to ten methionine residues.Likewise, the negative ion LC-ESI data shows that the predominantHMB-(Met)n co-oligomers contain one HMB residue and four to ninemethionine residues. The relative distribution of (Met)n oligomers andHMB-(Met)n co-oligomers presented in FIGS. 26 and 27.

Example 5 Synthesis of HMB-(Met)n Co-Oligomers with HMB-Methyl Ester andMet-Ethyl Ester

ESI-MS Results

Further experiments were conducted to confirm that HMB is attached atthe N-terminal of the oligomers chain. In one such experiment methylester of HMB and ethyl ester of methionine were prepared. Equivimolaramounts of mixed esters were subjected to papain mediatedoligomerization and co-oligomerization at pH 5.5 with the procedureoutlined in Example 1. The product was washed with water until it wasfree of monomers. The product was then freeze-dried, dissolved in DMSOand introduced in the MS through an ESI interface. The positive andnegative ion spectra obtained for the mixed co-oligomers are shown inFIGS. 28A and 28B respectively.

The positive ion spectra (FIG. 28A) of shows two series of ions that are28 mass units apart. One series containing ions m/z 674, 805, 936, 1067,1198 and 1229 correspond to ((Met)_(n)+H⁺) ions. The other series ofions which occur at m/z 702, 833, 964, 1095, 1226 and 1357 correspond to((Met)n-OET)+H⁺ ions. Ions in both series are 131 mass units or onemethionine residue (C₅H₉NOS) apart. In both series, the value of n liesbetween 4-10.

Incorporation of HMB at the C-terminal end of the polymethionine chainshould have resulted in a series of ions corresponding to((Met)n-HMB-OCH₃)+H⁺ ions. Such ions, if formed, would appear at m/z688, 819, 958, 1081, 1212 and 1343. However, none of the peaks of thisseries were observed in the spectra. Similar results were obtained inthe case of the negative ions (FIG. 28B). The absence of the methylgroup provides indirect evidence that HMB is incorporated at theN-terminal end. It should be pointed out that dominant ions obtained innegative ion mode were adduct ions and contained a dimethyl sulfonemoiety.

Example 6 Sonic Spray—MS-MS Results

The polymethionine and HMB-polymethionine prepared from Met-ethyl esterand HMB-ethyl ester were also subjected to MS-MS experiments. Thefreeze-dried precipitates were dissolved in DMSO (2 μg/μl) and thesolution was introduced into the mass spectrometer with the sonic sprayinterface at the rate of 1 mL/hr. The solution was mixed with 1:1acetonitrile:water mixture containing 0.1% acetic acid. The total fluidvolume entering the SSI-MS was maintained at 0.2 mL/min. The parent ionand spectra obtained with the system are shown in FIG. 29.

Ions of (Met)n-O Et+H⁺ corresponding to methionine hexamer, heptamer,octamer and nanomer were observed at m/z 833, 964, 1095 and 1225 amurespectively. For MS-MS experiment, the ion at m/z 833 was excited withan auxiliary Vrf and subjected to collision induced dissociation. Thedaughter spectrum of (Met)₆-EE is shown in FIG. 30. The prominentfragment ion was observed at m/z 657, this ion results from the cleavageof the amide bond resulting in the loss of Met-O Et (C₇H₁₄NO₂S) moietyfrom the C-terminal end. Similar results were obtained with molecularions resulting from HMB-(Met)_(n) ⁻. Daughter ions resulting from theloss of HMB-O Methyl (C₆H₁₂NO₂S) moiety were not observed, indicatingthe absence of HMB-O Methyl at the C-terminal end.

Example 7 Papain Catalyzed Synthesis of HMB-Tyrosine Co-Oligomers

Synthesis of tyrosine oligomers and HMB-tyrosine co-oligomers wasinitiated with tyrosine ethyl ester (Tyr-OMe) and HMB ethyl ester as themonomer substrate. The overall synthesis and purification approach wassimilar to the one used for methionine and HMB-methionine described inExample 1.

Tyr-OMe (equal amounts (wt %) in the case of HMB-OEt and Tyr-OMe) weredissolved in 9.5 mL of 1M citrate buffer. EDTA and L-Cysteine wereadded. The reaction mixture was adjusted to a pH of 5.5 and 0.5 mL ofpapain suspension was added to the mixture. The reaction mixture wasincubated in a shaker for 24 hours. The enzyme was then denatured byheating the reaction mixture to 80 EC for 10 minutes. The reactionmixture was cooled to room temperature.

The reaction mixture was filtered to collect the precipitate. Theoligomer and co-oligomers in the precipitate were dissolved in DMSO toseparate them from the monomers which are relatively insoluble in thesolvent. The separated solvent was then evaporated to re-precipitate theoligomer and co-oligomers, which were then washed with water andfreeze-dried.

The recipe for the tyrosine oligomers and HMB-tyrosine co-oligomersexperiment are provided in Table 4.

Table 4 Reaction Mixtures Used for Tyrosine Oligomerization andHMB-Tyrosine Co-Oligomerization Components MW Moles Wt

Components MW Moles Wt AA-ester 3 g L-Cys•HCl•H₂O 175.6 100 mM 0.1756 gEDTA (anhyd) 292.0 10 mM 0.0292 g Na Citrate 294.1 1 M 2.941 Papain21428D 7*10⁻⁵ M 15 mg Volume 10 mL pH 5.5

The reaction achieved a reaction rate similar to that observed withmethionine in Example 1. Approximate oligomer yield was 70-80%. Thefreeze-dried oligomer precipitates were solubilized in DMSO. Thesolution concentration was brought to approximately 2 μg/μl. Thesolution was mixed with 1:1 acetonitrile:water mixture containing 0.1%acetic acid. The total fluid volume entering the ESI-MS was maintainedat 0.2 mL min.⁻¹ The positive mass spectrum of the tyrosine oligomers isshown in FIG. 31.

Two sets of prominent ions appeared in the Tyr oligomers spectra. Oneseries of ions appeared at m/z 834, 997, 1160 and 1323, while the otherseries of ions were found at m/z 862, 1025, 1188 and 1351. The ions inthe first series represent (Tyr)n+H⁺, while the ions in the secondseries represent (Tyr)n-OEt+H⁺. The ions in the two series are 28 amu(C₂H₄) apart indicating the presence of O-Et at the C-terminal end inone series. Ions within the two series are separated by 163 amu,corresponding to the repeating unit of the Tyr residue (C₉H₉NO₂). Thus,the protonated ion at 862 most probably represents the tyrosineoligomers with five residues and an ethyl ester attached to theC-terminal end. Similarly, the ion at m/z 1025 most likely results fromthe (Tyr)₆.OEt+H⁺. The ion at m/z 997 results from (Tyr)₆+H⁺. Thepresence of oligomers with 5 to 8 Tyr residues is clearly evident,furthermore, the (Tyr)₆ was found to be the most prominent oligomer.

The positive ion mass spectrum of HMB-tyrosine co-oligomer is shown inFIG. 32A. The dominant ions in this spectrum were the same ions observedin the positive ion spectrum of polytyrosines, FIG. 31A. However,additional ions appeared at m/z 831, 994 and 1157. These ions appear ata mass difference of 133, suggesting the presence of a HMB residue inthe co-oligomer. The peak at m/z 831 most probably represents theco-oligomer with one HMB residue and 4 tyrosine residues with the ethylester moiety (HMB-(Tyr)₄ OEt+H⁺). Similarly, the residues at m/z 994 and1157 represent co-oligomers with one HMB residue and 6 and 7 tyrosineresidues respectively. The weak intensity of these ions in part relateto lower proton affinity of the hydroxyl group.

Example 8 Papain Catalyzed Synthesis of HMB-Leucine Co-Oligomers

The papain-catalyzed synthesis of leucine and HMB co-oligomers wasperformed. Synthesis of leucine oligomers and HMB-leucine co-oligomerswas initiated with leucine ethyl ester and HMB ethyl ester as thesubstrates. The overall synthesis and purification approach was similarto the one used in the case of methionine and HMB-methionine. Reactionrates similar to those obtained with methionine and tyrosine wereachieved. Approximate oligomer yield was 58%. The freeze-dried oligomerprecipitates were solubilized in DMSO. The solution concentration wasbrought to approximately 2 μg/μl. The solution was mixed with 1:1acetonitrile:water mixture containing 0.1% acetic acid. The total fluidvolume entering the ESI-MS was maintained at 0.2 mL min.⁻¹ The positivemass spectrum of the leucine oligomers is shown in FIG. 33A.

Four sets of ions appeared in the positive ion spectra of (Leu)_(n). Oneset of ions corresponding to (Leu)_(n)+H⁺ appeared at m/z 698, 811 and924. The other set of ions appeared at m/z 720, 833 and 947 andcorrespond to (Leu)_(n)+Na⁺. Another set of ions appeared at m/z 747,860 and 973 which correspond to (Leu)₆-OEt +Na⁺, (Leu)₇-OEt+Na⁺ and(Leu)₈-OEt+Na⁺. However, the two prominent ions in the spectra appear tobe (Leu)₆-OEt Na+Na⁺ and (Leu)₇-OEt Na+Na⁺. These results clearly showthat the dominant oligomers are (Leu)₅, (Leu)₆, (Leu)₇ and (Leu)₈. Themass difference of 28 amu (C₂H₄) indicates the presence of O-Et at theC-terminal. Ions within the two series are separated by 113 amu,corresponding to the repeating unit of the Leu residue (C₆H₁₁NO). Thus,the doubly sodiated (Na₂) ion at m/z 770 and 883 most probablyrepresents leucine oligomers with six and seven residues and a ethylester attached to the C-terminal end.

The negative ion mass spectrum of the leucine oligomers is shown in FIG.33B. The overall appearance of the spectra is similar to that of thepositive ion spectra. The two dominant ion in this spectra correspond to(Leu)₆-OEt+Na and (Leu)₇-OEt+Na.

The positive and the negative ion spectra of HMB-Leu co-oligomers areshown in FIG. 34. As expected, the positive spectra contained all of thedominant ions observed in the positive ion ESI-MS spectra of (Leu)_(n).However, three additional strong ions at m/z 740, 853 and 866 were alsofound. These masses correspond to sodiated co-oligomers HMB-(Leu)₅+Na⁺,HMB-(Leu)₆+Na⁺ and HMB-(Leu)₇+Na⁺ respectively. Thus, formation ofco-oligomers with one HMB residue with five to seven leucine residues isclearly evident.

Example 9 Papain Catalyzed Synthesis of HMB-Phenylalanine Co-Oligomers

Papain catalyzed synthesis of phenylalanine and HMB co-oligomers wasalso conducted. Synthesis of phenylalanine oligomers and HMB-phenylalanine co-oligomers was initiated with phenylalanine ethyl esterand HMB ethyl ester as the substrates. The overall synthesis andpurification approach was similar to the one used in the case ofmethionine and HMB-methionine in Example 1. The oligomerization reactiondid not proceed when phenylalanine was the only substrate present in thereaction mixture. The reaction did proceed when HMB-ethyl ester wasadded to the reaction mixture. Reaction rates similar to those withmethionine and tyrosine were achieved. Approximate oligomer yield was90%. The freeze-dried oligomer precipitates were solubilized in DMSO.The solution concentration was brought to approximately 2 μg/μl. Thesolution was mixed with 1:1 acetonitrile:water mixture containing 0.1%acetic acid. The total fluid volume entering the ESI-MS was maintainedat 0.2 mL min.⁻¹ As stated earlier, phenylalanine homo-oligomers werenot formed.

The ESI-MS results of HMB-PheHMB co-oligomerization reaction are givenin FIG. 35. The positive ion spectra of HMB-Phe co-oligomers aredepicted in FIG. 35A, while the negative ion spectra are depicted inFIG. 35B. The positive spectra of the co-oligomers yielded three ionpeaks at m/z 790, 937 and 1084. The mass difference between these ionsis 147 or a difference of one phenylalanine residue (C₆H₆NO=147). Them/z values of the ions most likely correspond to HMB-(Phe)₄-OEt +Na⁺,HMB-(Phe)₅-OEt+Na⁺ and HMB-(Phe)₆-OEt+Na⁺. Thus, formation ofco-oligomers with one HMB residue and four to six Phe residues isclearly evident.

Example 10 Optimization of (Lys)n Oligomers and HMB-(Lys)n Co-OligomersSynthesis

Experiments were conducted to optimize the reaction conditions forpapain catalyzed synthesis of lysine oligomers and lysine co-oligomerswith HMB. Reactions were carried out in two systems. The first systemconsisted of an aqueous phase and an immiscible organic phase, while thesecond system comprised a three-phase system consisting of an aqueousphase sandwiched between two mutually immiscible organic phases.

-   A. Two Phase Reaction System

The two-phase reaction system consisted of a small amount of polar phaseand a larger amount of an immiscible non-polar phase. The polar phasecomprised water, isopropyl amino ethyl and mercaptoethanol. This phasealso contained the amino acid ester substrate and papain. Duringoptimization, parameters such as the volume ratio of the aqueous and thenon-aqueous phase, composition of additives, concentrations of theadditives, concentrations of the substrates, and the concentration ofthe enzyme were varied. The effect of these parameters on the degree ofoligomerization and yield were monitored. The results of the experimentsare summarized below:

-   A.1 Aqueous: Organic Phase Ratio

To optimize the volume ratio of the aqueous and organic phases(toluene), the oligomer yields and degree of oligomerization weremonitored over phase ratios ranging from 1:9-1:99, the reaction wasallowed to proceed for 24 hours. Oligomer were recovered from theaqueous phase and analyzed. Results of these experiments are shown inFIG. 36.

The results indicate that the yields dropped at ratios below 19 and thehigher ratios did not lead to an appreciable change in total yield. Theresults also showed that while the total yield did not change at higherorganic solvent volumes, the degree of oligomerization was affected.Higher toluene volume led to a decrease in the degree ofoligomerization. The length of oligomers chains at phase ratio 1:19extended up to nine lysine residues (Lys)₉, whereas at phase ratio 1:39,the largest oligomers contained only six lysine residues (Lys)₆, FIG.37. In light of these results and to conserving organic solvent, allsubsequent experiments were carried out at phase ratio 1:19.

-   A.2 Optimization of Additive Concentration

The effect of the concentration of additives (mercaptoethanol andisopropyl ethyl amine) on yield and degree of oligomerization was alsoexamined. These additives act as antioxidants and prevent oxidation ofcysteine moiety in the enzyme. The reaction was carried out for 24hours. Oligomers were recovered from the aqueous phase and analyzed.Results showed that concentration of additives had a marked effect onthe total yield and the degree of oligomerization. The total yieldincreased with an increase in additive concentration from 0.1-2%.However, a pronounced decrease in total oligomers yield was observedwhen the additive concentration was increased above 5%. A 2% additiveconcentration was found to the optimum under conditions used in theseexperiments. The total oligomers yield at this additive concentrationwas approximately 87%, FIG. 38.

The degree of oligomerization was found to increase with an increase inadditive concentration up to 2%, still higher concentrations did notlead an appreciable change in the oligomers distribution as shown inFIG. 39.

-   A.3 Optimization of Substrate Concentration

A series of experiments were carried to optimize the substrate (lysineethyl ester) concentration at a fixed enzyme activity. The concentrationof substrate was varied five folds, from 500 mM to 2,500 mM, while theenzyme concentration was held constant at 1.21 mM. Oligomerizationreactions were allowed to proceed for 24 hours after which the enzymewas deactivated and oligomers recovered from the aqueous phase andanalyzed. A plot of the percent oligomers yield (total oligomersmass/total substrate mass×100) vs the substrate mass is shown in FIG.40.

The results show that the highest conversion efficiency was achieved ata substrate concentration of 1000 mM. A noticeable drop in conversionefficiency above this concentration was clearly evident. The degree ofoligomerization was also affected by the substrate concentration. Ingeneral, higher concentration led to the formation of oligomers withhigher lysine residues (e.g., the most abundant oligomers at 500 mMlysine ethyl ester was (Lys)₄ and the yield of higher homologs was quitelow). The most abundant oligomer was (Lys)₅. In addition, concentrationsof higher homologs (Lys)₆, (Lys)₇ and (Lys)₈ were noticeably higher,FIG. 41.

-   A.4 Optimization of Incubation Period

Another set of experiments was carried out to determine an optimumincubation period for oligomerization of lysine. The reaction wereconducted with 1:19 phase ratio, 1 M substrate concentration and 1%additive concentration. The reaction was allowed to continue for timeperiods ranging between 30 minutes to 24 hours. After each time period,the reaction was brought to halt by deactivating the enzyme. Theoligomers were recovered from the aqueous phase and analyzed. The totaloligomers yield obtained at various time periods is shown in FIG. 42.

The graph shows that the reaction is nearly complete within the firstfour hours and only a marginal increase in yield is obtained at longerincubation periods. Analyses of oligomers obtained after different timeperiods showed that the time periods shorter than 4 hours yield an evendistribution of oligomers from (Lys)₂ to (Lyn)₈, while the longerperiods yield higher concentrations of (Lyn)₄ to (Lys)₆ oligomers, FIG.43.

-   B. Three Phase System

The three-phase system consisted of an aqueous phase present between twoimmiscible non-aqueous phases, one lighter than the aqueous phase andthe other heavier than the aqueous phase. The heavier phase compriseddecafluoropentane and the lighter phase comprised n-octane. Isopropylethyl amine and mercaptoethanol additives were added to the aqueousphase along with the lysine ethyl ester (substrate) and papain (enzyme).The effects of parameters such as the relative volumes of aqueous tonon-aqueous phases, the concentration of the additives, the substrateconcentration and the enzyme activity on oligomers yield and degree ofoligomerization were monitored through a set of experiments.

-   B1. Optimization of Aqueous and Non-Aqueous Phase Ratio

The ratio of the non-aqueous phase volume to aqueous phase volume wasvaried by changing the volumes of the two organic solvents in equalproportion while holding the aqueous phase volume constant. Substrates,antioxidant additives and enzyme were added to the aqueous phase. Thereactants and the enzyme were placed in a stirred reactor and allowed toincubate at 37° C. for 24 hours. The total oligomers yield wasdetermined gravimetrically, while the degree of oligomerization wasdetermined through RPLC analysis. Results of gravimetric determinationare represented in FIG. 44.

The results show that the oligomer yield increased with an increase intotal organic phase. However, the increase in yield was relatively smallabove an aqueous: organic ratio of 1:10. The effect of phase ratio onthe degree of oligomerization is shown in FIG. 45. Results show thatwhile the total yields are lower (approximately 15-50%) at the lowerphase ratios, the degree of oligomerization is higher and oligomers withup to 10 lysine residues can be readily obtained. At higher phaseratios, the total oligomers yields are significantly greater (e.g., upto approximately 85%). The degree of oligomerization was generallylower, however, as the predominant oligomers formed under theseconditions contained three to five lysine residues.

-   B2. Optimization of Additives Concentration

The effect of the total additive concentration on the oligomers yieldand degree of oligomerization was examined. At low additiveconcentrations (e.g., concentrations <1.5%), the overall oligomersyields were low (e.g., approximately 40%). An increase in additiveconcentration up to 2.5% led to an increase in the oligomers yield,however, concentration above this level did not lead to higher yields,FIG. 46. The concentration of the additives did not assert a pronouncedeffect on the distribution of lysine oligomers, FIG. 47.

-   B3. Optimization of Incubation Period

The incubation period for the three-phase reaction system was examinedthrough another set of experiments. The experiments were conducted withthree phase reaction mixtures consisting of aqueous phase and totalorganic phases, the aqueous:organic phase ratio was set at 1:9. Theadditive concentrations were varied. The incubation periods were variedfrom 30 minutes to 30 hours. After each time period, total oligomeryield and degree of oligomerization were examined. Results are shown inFIG. 48. The results indicate that the reaction reaches completion inapproximately six hours and nearly 90% of the initial substrate mass isconverted into the oligomers. Longer incubation periods did not lead tohigher yields.

RPLC results showed that the longer incubation periods favored oligomerswith smaller lysine residues. The predominant residue after a 24 hourincubation period was found to be (Lys)₄, FIG. 50.

The studies above demonstrate the effect of various parameters in thethree phase and two-phase systems and can be used to tailor the reactionto produce the required residue range and composition.

Example 11 Synthesis of Tryptophan Oligomers and HMB-TryptophanCo-Oligomers

A procedure similar to one used for the synthesis of Met oligomers andHMB-Met co-oligomers of Example 1 was employed for the synthesis oftryptophan oligomers and HMB-Tryptophan co-oligomers.

Trp-OMe was dissolved in 4.5 mL of 2M citrate buffer along with the EDTAand L-Cysteine. The pH was adjusted to 5 and 0.5 mL of papain suspensionwas added to the mixture. The mixture was placed in a shaker for threedays and incubated. After three days, the enzyme was denatured byheating the broth for a duration of 10 minutes at 80° C.

The broth was filtered to collect the precipitate. Alternatively, thebroth could be centrifuged to collect the precipitate. The oligomer andco-oligomer precipitate was dissolved in DMF to separate them from themonomers which are relatively insoluble in the solvent. The solvent wasevaporated and the precipitate was washed with water, followed byfreeze-drying the precipitate to obtain the dry oligomers andco-oligomers.

The procedure was also performed wherein L-Tyrosine Ethyl Ester wassubstituted for Trp-OMe. The recipes for synthesis of differentoligomers and co-oligomers are summarized below in Table 5. TABLE 5Recipes for the Synthesis and Purification of L-Trp Homo-Oligomers andHMB-L-Trp Hetero-Co-Oligomers Jost/Trp-OEt Selvi/Trp-OMe Components MWMoles Wt Moles Wt Wt AA-ester 3 g 0.38 M 370 mg L-Cys•HCl•H₂O 175.6 100mM 0.1756 g 100 mM 0.0878 EDTA (anhyd) 292.0 10 mM 0.0292 g 10 mM0.01461 g Na Citrate 294.1 1 M 2.941 2 M 2.941 g Papain 21428D 7*10⁻⁵ M15 mg 1.4*10⁻⁴ M 15 mg Volume 10 mL 5 mL pH 5.5 5

Example 12 Synthesis and Purification of Leucine, Phenylalanine, andTryptophan Oligomers

A procedure similar to one used for the synthesis of Met oligomers andHMB-Met co-oligomers of Example 1 was employed for the synthesis andpurification of leucine, phenylalanine, and tryptophan oligomers. Anamino acid ester (e.g., 1 mmole) was dissolved in 10 mL of 2M phosphatebuffer solution at pH 7.5 containing 1 mM dithiothreithol and 5 mM EDTA.A papain suspension (e.g., 0.1 mmole) was added to the solution. Thesolution was incubated for two days with continuous shaking, after whichthe precipitate formed was filtered and washed with water several timesto remove the free monomers. The precipitate was dried in vacuo and thensubjected to analysis.

The recipes for synthesis of different oligomers are summarized below inTable 6. TABLE 6 Recipes for the Synthesis and Purification of Leucine,Phenylalanine, and Tryptophan Oligomers Components Moles MW WtAA-OEt•HCl 0.1 mM Dithiothreithol 1 mM 154.2 0.00152 g EDTA 5 mM 292.20.01461 g Na₂HPO₄/NaH₂PO₄ 2 M (Phosphate Buffer) Volume 10 mL 10 mL

Example 13 A General Procedure for the Synthesis and Purification ofOligomers

L-amino acid ethyl ester or D, L-amino acid esters and racemic HMB ethylester were dissolved in buffer containing L-cysteine, EDTA and sodiumcitrate at pH 5.5 as detailed in Tables 5-9. The buffer pH was set at5.5 and 0.5 mL of papain suspension containing 15 mg of protein wasadded to the reaction broth. After incubation in a shaker for 24 hoursat 35° C., the enzyme was denatured by heating the broth to 80° C. for10 minutes. The broth with denatured enzyme was then cooled to roomtemperature. The oligomer and co-oligomer precipitate obtained from thereaction was washed exhaustively with water to remove the monomers andthe washed precipitate was then freeze-dried. The freeze-dried oligomerand co-oligomer precipitate was solubilized in DMSO to form a 2 μg/μLsolution. To remove traces of free HMB-ester, HMB co-oligomers werere-precipitated by addition of distilled deionized water. The purifiedoligomers and co-oligomers were freeze dried. The oligomer andco-oligomer were dissolved in appropriate solvents or mixtures solution(DMSO and 1:1 acetonitrile: water mixture) for chemical characterizationwith liquid chromatography (LC) diode array detector, LC-electrospraymass spectrometry (ESI-MS), sonic spray ionization-MS (SSI-MS) andmatrix assisted desorption ionization-MS (MALDI-TOF).

Recipes for synthesis of different oligomers and co-oligomers aresummarized in Tables 7-11. TABLE 7 Recipes for the Synthesis andPurification of L-Met Oligomers and HMB-L-Met Co-Oligomers Components MWMoles Met HMB-Met L-AA- OEt•HCl 213.7 3 g 1.5 g DL-HMB-OEt 178 — 1.5 gL-Cys•HCl•H₂O 175.6 100 mM 0.1756 g 0.1756 g EDTA (anhyd) 292.0 10 mM0.0292 g 0.0292 g Na Citrate 294.1 1 M 2.941 g 2.941 g Papain 21428D7*10⁻⁵ M 15 mg 15 mg Volume 10 mL 10 mL pH 5.5 5.5

TABLE 8 Recipes for the Synthesis and Purification of L-Tyr Oligomersand HMB-L-Tyr Co-Oligomers Components MW Moles Tyr HMB-Tyr L-AA-OEt933.36 mg 466.82 mg DL-HMB-OEt 178 — 338.2 mg L-Cys•HCl•H₂O 175.6 100 mM0.1756 g 0.1756 g EDTA (anhyd) 292.0 10 mM 0.0292 g 0.0292 g Na Citrate294.1 1 M 2.941 g 2.941 g Papain 21428D 7*10⁻⁵ M 15 mg 15 mg Volume 10mL 10 mL pH 5.5 5.5

TABLE 9 Recipes for the Synthesis and Purification of L-Leu Oligomersand HMB-L-Leu Co-Oligomers Components MW Moles Leu HMB-Leu L-AA-Oet2.739 g 1.369 g DL-HMB-OEt 178 — 1.253 g L-Cys•HCl•H₂O 175.6 100 mM0.1756 g 0.1756 g EDTA (anhyd) 292.0 10 mM 0.0292 g 0.0292 g Na Citrate294.1 1 M 2.941 g 2.941 g Papain 21428D 7*10⁻⁵ M 15 mg 15 mg Volume 10mL 10 mL pH 5.5 5.5

TABLE 10 Recipes for the Synthesis and Purification of L-Phe Oligomersand HMB-L-Phe Co-Oligomers Components MW Moles Phe HMB-Phe L-AA-Oet1.1414 g 0.5707 g DL-HMB-OEt 178 — 0.4067 g L-Cys•HCl•H₂O 175.6 100 mM0.1756 g 0.1756 g EDTA (anhyd) 292.0 10 mM 0.0292 g 0.0292 g Na Citrate294.1 1 M 2.941 g 2.941 g Papain 21428D 7*10⁻⁵ M 15 mg 15 mg Volume 10mL 10 mL pH 5.5 5.5

TABLE 11 Recipes for the Synthesis and Purification of L-Trp Oligomersand HMB-L-Trp Co-Oligomers Components MW Moles Trp HMB-Trp L-AA-Oet 0.480.480 g DL-HMB-OEt 178 — 0.480 g L-Cys•HCl•H₂O 175.6 100 mM 0.1756 g0.1756 g EDTA (anhyd) 292.0 10 mM 0.0292 g 0.0292 g Na Citrate 294.1 1 M2.941 g 2.941 g Papain 21428D 7*10⁻⁵ M 15 mg 15 mg Volume 10 mL 10 mL pH5.5 5.5

Example 14 Enantioselectivity of Papain Catalyzed Oligomerization andCo-Oligomerization

A set of experiments was carried out to discern enantioselectivity ofpapain wherein oligomers and co-oligomers were catalyzed from an aminoacid and HMB. The experiments entailed enantioselective determination ofthe reactants (e.g., methionine and HMB co-oligomerization fromenantiomeric mixtures of D, L-methionine and D, L-HMB and separation ofsupernatant and oligomer and co-oligomer precipitates). The supernatantwas filtered to remove suspended matter, and the precipitate waspurified through repeated washing and DMSO back-extraction. The purifiedoligomer and co-oligomer precipitates were hydrolyzed with acid. Thereactant solutions, supernatant, and hydrolyzates were subjected toenantioselective HPLC analysis. The results of the experiments indicatedthat catalytic co-oligomerization of amino acids and HMB isenantioselective wherein only the L-HMB and the L-amino acid isomersundergo oligomerization and co-oligomerization.

Papain Catalyzed Synthesis

The Met oligomers and HMB-Met co-oligomers were synthesized throughpapain mediated enzymatic reactions at pH 5.5. The synthesis involvedthe following steps:

-   -   Racemic mixtures of both D, L-Met-OEt and D, L-HMB-OEt were        dissolved in 4.5 mL of 2M citrate buffer along with EDTA and        L-Cysteine. The pH was set to 5 and 0.5 mL of papain suspension        was added to the mixture. The mixture was incubated in a shaker        for 3 days.    -   After the mixture was incubated in a shaker for 3 days, the        enzyme (e.g., papain) was denatured by heating the mixture for        10 minutes at 80° C.    -   The mixture was filtered and the precipitate collected.        (Alternatively, the mixture may be centrifuged to separate the        precipitate from the supernatant).    -   The oligomers and co-oligomers were dissolved in DMF to separate        them from the monomers, which are relatively insoluble in DMF.

The solvent was evaporated and the precipitate washed with water,followed by freeze drying to obtain the dry oligomers and co-oligomers.

The recipe for synthesis of oligomers of α-amino acid isomers havingidentical chiral configurations and co-oligomers formed from HMB isomershaving a specific chiral configuration and α-amino acid isomers havingidentical chiral configurations is summarized below in Table 12. TABLE12 Recipe for the Synthesis and Purification of L-Met Oligomers andL-HMB-L-Met Co-Oligomers From Racemic Mixtures of D,L-MetOEt andD,L-HMB-OEt MetOEt/HMB-OEt Components MW Moles Wt AA-ester 3 gL-Cys•HCl•H₂O 175.6 100 mM 0.1756 g EDTA (anhyd) 292.0 10 mM 0.0292 g NaCitrate 294.1 1 M 2.941 Papain 21428D 7*10⁻⁵ M 15 mg Volume 10 mL pH 5.5Hydrolysis of Met Oligomers and Met/HMB Co-Oligomers

A 25 mg aliquot of purified oligomers and co-oligomers obtained from theracemic Met-OEt and HMB-OEt was placed in 10 mL vials and hydrolyzedwith 2 mL of 6.05(N) HCl at 110° C. for 24 hours. The clear solutionsobtained after hydrolysis were diluted with nanopure water and injectedin an LC equipped a diode array detector (DAD).

Enantioselective HPLC

Enantioselective HPLC analysis was carried out with a proline-Cu basedcolumn EC 250/4 Nucleosil Chiral 1 (Macherey-Nagel, Inc., Easton, Pa.).The mobile phase consisted of 0.5 mM cupric sulfate (pentahydrate)solution in nanopure water. Column oven temperature was maintained at60° C. Separated analytes were monitored with a UV absorbance DAD.

Results

The HPLC results showed that the Met-OEt and HMB-OEt reactants wereracemic mixtures that contained equal amounts of D- and L-Met ethylester enantiomers and D- and L-HMB ethyl ester enantiomers. See FIGS. 51and 52. Following oligomerization and co-oligomerization and separationof the precipitates, the supernatant was analyzed and found to containsignificantly higher concentrations of D-Met and D-HMB than L-Met andL-HMB. The relative concentrations of D-Met and L-Met were found to beapproximately 97%:<2.5% respectively. The relative concentrations ofD-HMB and L-HMB were found to be approximately 75% and 25% respectively.

The selective oligomerization and co-oligomerization of L-Met and L-HMBwere observed in the results from the enantioselective HPLC analysis.The analysis indicated that the oligomer and co-oligomer hydrolyzate wasfound to contain only the L-Met and the L-HMB isomers. See FIGS. 53 and54. While FIG. 54 indicates the presence of a D-HMB isomer peak, whenthe precipitate was washed, dissolved in DMSO, and reprecipitated, theD-HMB isomer peak disappeared, indicating that the D-HMB present wasinitially absorbed to the oligomer and co-oligomer precipitate, but wasnot covalently bonded within the oligomer and co-oligomer precipitate.

Example 15 Papain Catalyzed Synthesis of Lactic Acid-Amino AcidOligomers

This example demonstrates the synthesis of co-oligomers comprisinglactic acid (an α-hydroxy carboxylic acid) with oligomers of the α-aminoacids methionine, leucine, tyrosine, phenylalanine and tryptophan.

The experiment consisted of esterifying D,L-Lactic acid with acidifiedethyl alcohol by refluxing it at 80° C. for 8 hrs to produce lactic acidethyl ester, which was used in each of the oligomerization reactions.The oligomerization consisted of forming a mixture by dissolving each ofthe amino acid ethyl esters and the lactic acid ethyl ester in variousamounts in a pH 5.5 buffer containing, L-cysteine, EDTA and sodiumcitrate as shown in the tables below. At the end of 24 hours, themixture was heated to 80° C. for 10 mins to denature the enzyme. Thesupernatant was analyzed on RPLC to determine the yield of the reaction.The precipitate was washed thoroughly with water to obtain a monomer(amino acid and lactic acid) free product. The product was freeze driedand a small part of it was dissolved in DMSO and introduced into theESI-MS and the mass spectrum obtained was recorded.

-   1. Lactic Acid-Methionine Co-Oligomerization

Lactic acid-methionine oligomers were synthesized using the generalprocedure described above. The ingredients for the oligomerizationreaction mixture consisted of the following: Composition L-MetEE-HCI (g)1.5 LAEE (g) 1.5 L-Cysteine (mg) 175.6 EDTA (mg) 29.2 Sodium Citrate (g)2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

The oligomerization produced an yield of 75% and the positive ion andnegative ion spectra are reproduced in FIGS. 55A and 55B respectively.The positive ion spectrum shows the presence of 2 dominant peaks at 834and 965. These peaks most probably represent the homo-oligomers,^(N)Met-(Met)₄-Met^(C)-OEt and ^(N)Net-(Met)₅-Met^(C)-OEt respectivelywhich are separated by the repeating methionine residue unit of mass131.2.

The negative ion spectrum shows the presence of a series of peaks eachseparated by around 131 mass units. One set of peaks appear at 774 and905 and these most probably represent the deprotonated ions,^(N)LA-(Met)₄-Met^(C)-OEt and ^(N)LA-(Met)₅-Met^(C)-OEt respectively.Another set of ions, appear at 809 and 940 and these mot probablyrepresent the adducts of the above co-oligomers with the chloride ion.

-   2. Lactic Acid-Tyrosine Co-Oligomerization

Lactic acid-tyrosine oligomers were synthesized using the generalprocedure described above. The ingredients for the oligomerizationreaction mixture consisted of the following: Composition L-TyrEE-HCI(mg) 466.8 LAEE (mg) 466.8 L-Cysteine (mg) 175.6 EDTA (mg) 29.2 SodiumCitrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

A yield of 98% with respect to tyrosine was obtained. The positive ionand negative ion spectra are provided in FIGS. 56A and 56B respectively.The positive ion spectra shows the presence of a evenly spaced sets oftwo peaks with each peak separated from the other by 28 amu. This massrepresents the difference in mass between C-terminal free acid and ethylester. Each set of peaks is separated by a mass of 163 units with is therepeating unit of the tyrosine residue. While one set appeared at 833,996, 1159 and 1322 most probably representing the protonatedhomo-oligomers of tyrosine namely, ^(N)Tyr-Tyr₃-Tyr^(C)-OH,^(N)Try-Tyr₄-Tyr^(C)-OH, ^(N)Tyr-Tyr₅-Tyr^(C)-OH and^(N)Tyr-Tyr₆-Tyr^(C)-OH, another set appeared 861, 1024 and 1187 andthese most probably represent the protonated forms of andNtyr-Tyr₃-Tyr^(C)-OEt, ^(N)Tyr-Tyr₄-Tyr^(C)-OEt andNtyr-Tyr₅-Tyr^(C)-OEt respectively. The negative ion spectrum reveals anumber of peaks with some of them forming a series separated by 163units. One set appears at 1096, 1259 and 1422 and these most probablyrepresent the presence of deprotonated co-oligomer ions formed from^(N)LA-Tyr₅-Tyr^(C)-OEt, ^(N)LA-Tyr₆-Tyr^(C)-OEt and^(N)LA-Tyr₇-Tyr^(C)-OEt respectively.

-   3. Lactic Acid-Leucine Co-Oligomerization

Lactic acid-leucine oligomers were synthesized using the generalprocedure described above. The ingredients for the oligomerizationreaction mixture consisted of the following: Composition L-LeuEE-HCI(mg) 684.5 LAEE (mg) 684.5 L-Cysteine (mg) 175.6 EDTA (mg) 29.2 SodiumCitrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

The oligomerization produced a yield of 40% with respect to leucine. Thepositive and negative ion specta are provided in FIGS. 57A and 57Brespectively. The positive ion spectrum has a pair of peaks at 726 and839 and these ions are separated by the repeating residue unit ofleucine (113 amu) and they most probably represent the protonated ionsof homo-oligomers, ^(N)Lleu-Leu₄-Leu^(C)-OEt and^(N)Leu-Leu₅-Leu^(C)-OEt respectively. Another ion appears at 698 andthis is most probably the protonated ion of the homo-oligomer^(N)Leu-Leu₄-Leu^(C)-OH. In addition to these peaks, another pair ofpeaks appear at 685 and 798 and these are again separated by therepeating unit of leucine and these most probably correspond to theprotonated forms of the co-oligomer peaks, ^(N)LA-Leu₄-Leu^(C)-OEt and^(N)LA-Leu₅-Leu^(C)-OEt respectively. The peaks corresponding to^(N)LA-Leu₄-Leu^(C)-OH+H⁺ and ^(N)LA-Leu₅-Leu^(C)-OEt+Na⁺ appear at 657and 821 respectively. The negative ion spectrum reveals a series ofpeaks each separated by 113 amu. These appear at 683, 796 and 909 andthey most probably represent the deprotonated forms of the co-oligomers^(N)LA-Leu₄-Leu^(C)-OEt-^(N)LA-Leu₅-Leu^(C)-OEt and^(N)LA-Leu₆-Leu^(C)-OEt respectively.

-   4. Lactic Acid-Tryptophan Co-Oligomerization

Lactic acid-tryptophan oligomers were synthesized using the generalprocedure described above. The ingredients for the oligomerizationreaction mixture consisted of the following: Composition L-TrpEE-HCI(mg) 480.0 LAEE (mg) 480.0 L-Cysteine (mg) 175.6 EDTA (mg) 29.2 SodiumCitrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

An oligomerization yield of 94% was obtained with respect to tryptophan.The positive ion and negative ion spectra are produced in FIGS. 58A and58B respectively. A series of ions separated b 186 units at 419, 605,791 and 977 appears in the positive ion spectrum. These indicate thepresence of the protonated homo-oligomers of the form^(N)Trp_(n)-Trp^(c)-OEt with n 1 to 5. The ^(N)Trp-Trp-Trp^(C)-OH+H⁺ ionappears at 577. The ion at 700 most probably represent the loneco-oligomer peak of ^(N)LA-Trp₂-Trp^(C)-OEt +Na⁺. The negative ionspectrum shows the presence of deprotonated ^(N)Trp-Trp₂-Trp^(C)-OEt asthe base peak at 789. However, neither of the spectra does showexplicitly show the presence of co-oligomer peaks. Further work withLC-MS needs to be done to separate the oligomer from the co-oligomersand prove the presence/absence of the co-oligomers.

-   5. Lactic Acid-Phenylalanine Co-Oligomerization

Lactic acid-phenylalanine oligomers were synthesized using the generalprocedure described above. The ingredients for the oligomerizationreaction mixture consisted of the following: Composition L-PheEE-HCI(mg) 570.7 LA-IPA (mg) 570.7 L-Cysteine (mg) 175.6 EDTA (mg) 29.2 SodiumCitrate (g) 2.9 Papain (mg) 30.0 Total Volume (ml) 10.0

The lactic acid was esterified with iso-propyl alcohol and was usedalong with the phenylalanine ethyl ester. An oligomerization yield of80% was obtained with respect to phenylalanine. The positive andnegative ion spectrum is provided in FIGS. 59A and 59B respectively. Thepositive ion spectrum reveals the strong presence of sodiatedco-oligomers ions of ^(N)LA-(Phe)₃-Phe^(C)-OEt+Na⁺ and^(N)LA-(Phe)₄-Phe^(C)-OEt+Na⁺ at masses 730 and 877 respectively. Thesepeaks are separated by 147 units, which is the repeating residue unit ofphenylalanine. The absence of any oligomer peaks is due to fact that thephenylaline does not oligomerize without a N-terminal starter molecule.This points to the presence of LA at the N-terminal end of the oligomer.The presence of ethyl ester (and absence of the iso-propyl ester) at theC-terminal end also reinforces this result. The negative ion spectrumreveals two sets of peaks with each set separated by 147 units. Thesesets represent the deprotonated co-oligomer peaks to form^(N)LA-Phe_(n)-Phe^(C)-OEt with n having values of 3 and 4.

In view of the above, it will be seen that the several objects of theinvention are achieved. As various changes could be made in the abovecompositions and methods without departing from the scope of theinvention, it is intended that all matter contained in the abovedescription be interpreted as illustrative and not in a limiting sense.

1. A process for the preparation of an oligomer, the process comprising:forming a reaction mixture comprising an enzyme, and an enantiomericmixture of an α-amino acid or a derivative thereof; and forming anoligomer from the reaction mixture, said oligomer incorporating one ofthe members of the enantiomeric mixture of the α-amino acid orderivative thereof in preference to the other member.
 2. A process asset forth in claim 1, wherein the enantiomeric mixture is a racemicmixture.
 3. A process as set forth in claim 1, wherein the oligomercomprises an α-amino acid or a derivative thereof comprising an oligomerof two or more α-amino acid monomers.
 4. A process as set forth in claim3, wherein the α-amino acid monomers comprise L-α-amino acid monomers.5. A process as set forth in claim 1, wherein the reaction mixturefurther comprises an α-hydroxy carboxylic acid or a derivative thereofand the formed oligomer comprises an oligomer of two or more α-aminoacid monomers bonded to a residue of the α-hydroxy carboxylic acid or aderivative thereof by an amide or ester linkage.
 6. A process as setforth in claim 5 wherein said α-hydroxy carboxylic acid or a derivativethereof is present in said reaction mixture in an enantiomeric mixture.7. A process as set forth in claim 6 wherein one α-hydroxy carboxylicacid enantiomer of the enantiomeric mixture is bonded to the oligomer inpreference to the other enantiomer of said enantiomeric mixture.
 8. Aprocess as set forth in claim 1 wherein the α-amino acid is selectedfrom the group consisting of methionine, lysine, or a derivativethereof.
 9. A process as set forth in claim 1 wherein said enantiomericmixture of α-amino acids comprises methionine and lysine.
 10. A processas set forth in claim 1 wherein the enzyme comprises a protease.
 11. Aprocess as set forth in claim 10 wherein the enzyme comprises a proteaseselected from the group consisting of papain, bromelain, cathepsin s,cathepsin b, capthesin c, and substilisin.
 12. A process as set forth inclaim 1 wherein the reaction mixture comprises an aqueous solution. 13.A process as set forth in claim 1 wherein the reaction mixture comprisesan aqueous phase and an organic solvent phase.
 14. A process as setforth in claim 1 wherein the reaction mixture comprises an α-hydroxycarboxylic acid derivative comprising a free acid, an acid halide, anamide, an ester or an anhydride.
 15. A process as set forth in claim 1wherein the reaction mixture comprises an α-amino acid derivativecomprising a free acid, an acid halide, an amide, an ester or ananhydride.
 16. A composition comprising a residue of an α-hydroxycarboxylic acid bonded to a peptide by an amide linkage, said peptidecomprising two or more α-amino acid residues, each of said α-amino acidsbeing independently selected from the group consisting of α-amino acids.17. A composition as set forth in claim 16 wherein more than 50% of theα-amino acid residues in the peptide are of identical chirality.
 18. Acomposition as set forth in claim 17 wherein essentially all of theα-amino acid residues in the peptide are of identical chirality.
 19. Acomposition comprising a residue of an α-hydroxy carboxylic acid bondedto a peptide by an ester linkage, said peptide comprising two or moreα-amino acid residues, each of said α-amino acids being independentlyselected from the group consisting of α-amino acids.
 20. A compositionas set forth in claim 19 wherein more than 50% of the α-amino acidresidues in the peptide are of identical chirality.
 21. A composition asset forth in claim 20 wherein essentially all of the α-amino acidresidues in the peptide are of identical chirality.
 22. A compositioncomprising:

wherein: R¹ is hydrogen, hydrocarbyl or substituted hydrocarbyl, R² ishydrogen, hydrocarbyl, substituted hydrocarbyl, or a hydroxy protectinggroup, R³ is hydrogen, hydrocarbyl or substituted hydrocarbyl, each AAis the residue of an α-amino acid or derivative thereof wherein greaterthan one-half of the AA residues are derived from α-amino acids orderivatives thereof having the an identical chiral configuration, and nis at least
 2. 23. The composition of claim 22 wherein R¹ isCH₃SCH₂CH₂—.
 24. The composition of claim 23 wherein R² is H.
 25. Thecomposition of claim 22 wherein R² is H.
 26. The composition of claim 22wherein n is at least 4 and no more than 12 and each AA is methionine.27. The composition of claim 22 wherein n is at least 3 and no more than5 and each AA is selected from the group consisting of methionine andlysine.
 28. The composition of claim 22 wherein n is at least 2 and nomore than 10 and each AA is selected from the group consisting ofmethionine and lysine.
 29. A process of providing a food ration to ananimal comprising providing the composition of claim 22 to the animalwherein the method of administration is selected from the groupconsisting of oral administration, eye spray, placement in ear,placement in nasal cavity, bucchal administration, sublingualadministration, rectal administration and injection.
 30. A process asset forth in claim 29 wherein the composition is orally administered tothe animals.
 31. A process as set forth in claim 30 wherein the animalis selected from the group consisting of ruminants, swine, poultry, andaquatic animals.
 32. A process as set forth in claim 31 wherein theruminant is a dairy cow or beef cattle.
 33. A process as set forth inclaim 32 wherein the cow is a lactating dairy cow.
 34. A process as setforth in claim 31 wherein the aquatic animal is a fish or crustacean.35. A process as set forth in claim 34 wherein the fish is selected fromthe group consisting of freshwater and salt water fish.
 36. A process asset forth in claim 35 wherein the freshwater and salt water fish areselected from the group consisting of carp, trout, catfish, bass, seabass, cod, and salmon.
 37. A process as set forth in claim 34 whereinthe crustaceans are selected from the group consisting of shrimp, crabs,lobster, and freshwater shrimp.
 38. A process for providing a foodration to an animal, the process comprising: providing an oligomer orco-oligomer composition prepared from a mixture containing an enzyme, anα-amino acid or a derivative thereof, and optionally, an α-hydroxycarboxylic acid or a derivative thereof, wherein the method ofadministration is selected from the group consisting of oraladministration, eye spray, placement in ear, placement in nasal cavity,bucchal administration, sublingual administration, rectal administrationand injection.
 39. A process as set forth in claim 38 wherein the animalis selected from the group consisting of ruminants, swine, poultry andaquatic animals.
 40. A process as set forth in claim 39 wherein theruminant is a dairy cow or beef cattle.
 41. A process as set forth inclaim 40 wherein the cow is a lactating dairy cow.
 42. A process as setforth in claim 39 wherein the aquatic animal is a fish or crustacean.43. A process as set forth in claim 42 wherein the fish is selected fromthe group consisting of freshwater and salt water fish.
 44. A process asset forth in claim 43 wherein the freshwater and salt water fish areselected from the group consisting of carp, trout, catfish, bass, seabass, cod, and salmon.
 45. A process as set forth in claim 42 whereinthe crustaceans are selected from the group consisting of shrimp, crabs,lobster, and freshwater shrimp.
 46. An orally administered dietarysupplement comprising a vitamin, a mineral or a nutrient coated with anoligomeric coating, said coating comprising a residue of an α-hydroxycarboxylic acid bonded to a peptide by an amide linkage, said peptidecomprising two or more α-amino acid residues, each of said α-amino acidsbeing independently selected from the group consisting of α-amino acids.47. A process for providing an animal with a dietary supplementcomprising a vitamin, mineral or nutrient, the process comprising:coating said vitamin, mineral or nutrient with a composition to form adietary supplement, said composition comprising a residue of anα-hydroxy carboxylic acid bonded to a peptide by an amide linkage, saidpeptide comprising two or more α-amino acid residues, each of saidα-amino acids being independently selected from the group consisting ofα-amino acids; and orally administering the dietary supplement to theanimal.
 48. A process as set forth in claim 47 wherein the peptidecomprises methionine.
 49. A process as set forth in claim 47 wherein theanimal is selected from the group consisting of ruminants, swine,poultry, and aquatic animals.
 50. A process as set forth in claim 49wherein the ruminant comprises a dairy cow or beef cattle.
 51. A processas set forth in claim 50 wherein the ruminant comprises a lactatingdairy cow.
 52. A process as set forth in claim 49 wherein the aquaticanimal comprises a fish or crustacean.
 53. A process as set forth inclaim 52 wherein the fish is selected from the group consisting offreshwater and salt water fish.
 54. A process as set forth in claim 53wherein the freshwater and salt water fish are selected from the groupconsisting of carp, trout, catfish, bass, sea bass, cod, and salmon. 55.A process as set forth in claim 52 wherein the crustaceans are selectedfrom the group consisting of shrimp, crabs, lobster, and freshwatershrimp.
 56. A process for providing an animal with a dietary supplementcomprising a vitamin, mineral or nutrient, the process comprising:preparing a mixture comprising an enzyme and at least one α-amino acid,each α-amino acid being present in the mixture as a free acid, acidhalide, amide, ester or anhydride independently of the other, forming anα-amino acid oligomer in the mixture, coating said vitamin, mineral ornutrient with the α-amino acid oligomer to form an α-amino acid oligomercoated dietary supplement, and orally administering the dietarysupplement to the animal.
 57. A process as set forth in claim 56 whereinthe animal comprises an aquatic animal selected from the groupconsisting of shrimp, carp, and trout.
 58. A process as set forth inclaim 57, wherein the α-amino acid oligomeric coating comprisesmethionine.
 59. A process for purifying an α-hydroxy carboxylic acidenantiomer or derivative thereof from an enantiomeric mixture, theprocess comprising: forming a reaction mixture comprising (i) anenantioselective enzyme, (ii) an enantiomeric mixture of an α-hydroxycarboxylic acid or a derivative thereof, and (iii) an enantiomericmixture of an α-amino acid or a derivative thereof; forming a reactionproduct from the reaction mixture, the reaction product comprising (i)an oligomer having a first α-hydroxy carboxylic acid enantiomer of theenantiomeric mixture incorporated therein in preference to the secondenantiomer, and (ii) unreacted second enantiomer; and separating theoligomer and unreacted second enantiomer from the reaction product andeach other.
 60. A process as set forth in claim 59, wherein theenantioselective enzyme promotes an esterification reaction.
 61. Aprocess as set forth in claim 60, wherein the enantioselective enzyme isa lipase enzyme.
 62. A process as set forth in claim 59, wherein thefirst enantiomer is recovered from the separated oligomer by hydrolyzingthe oligomer with acid and separating the first α-hydroxy carboxylicacid enantiomer or derivative thereof from other hydrolyzates.
 63. Aprocess as set forth in claim 62, wherein the first α-hydroxy carboxylicacid enantiomer or derivative thereof is separated by subjection toenantioselective chromatography.
 64. A process as set forth in claim 59,wherein the second unreacted α-hydroxy carboxylic acid enantiomer orderivative thereof is recovered from the reaction mixture by rotaryevaporation.
 65. A process as set forth in claim 59, wherein theα-hydroxy carboxylic acid comprises 2-hydroxy-4-(methylthio)butyric acidor a derivative thereof.
 66. A process as set forth in claim 59 whereinthe process further comprises recovering the first enantiomer from saidseparated oligomer by by hydrolyzing the oligomer with acid andseparating the first α-hydroxy carboxylic acid enantiomer or derivativethereof from other hydrolyzates; and converting a fraction of therecovered first enantiomer to the stereochemical form of the secondenantiomer thereby forming an enantiomeric mixture comprising the firstand second enantiomers.
 67. A process as set forth in claim 59 whereinthe process further comprises recovering the unreacted second enantiomerfrom the reaction product; and converting a fraction of the separatedunreacted second enantiomer to the stereochemical form of the firstenantiomer thereby forming an enantiomeric mixture comprising the firstand second enantiomers, at least a portion of the first enantiomer inthe enantiomeric mixture being derived from separated unreacted secondenantiomer.
 68. A process as set forth in claim 67 wherein theenantiomeric mixture formed from the recovered second enantiomer isrecycled for re-use in the formation of a reaction mixture.
 69. Aprocess as set forth in claim 67, wherein the enzyme is removed from thereaction mixture and recycled.
 70. A process as set forth in claim 69,wherein the enzyme is removed from the reaction mixture by sizeexclusion chromatography.
 71. A process as set forth in claim 67 whereinthe second unreacted α-amino acid enantiomers or derivatives thereof areseparated from the reaction mixture by rotary evaporation.
 72. A processfor purifying an enantiomeric mixture of α-amino acid or derivativethereof, the process comprising: forming a reaction mixture comprising(i) an enzyme, (ii) an enantiomeric mixture of α-amino acid or aderivative thereof, and (iii) an α-hydroxy carboxylic acid or aderivative thereof; forming a reaction product from the reactionmixture, the reaction product comprising (i) an oligomer incorporating afirst member of the enantiomeric mixture of α-amino acid or derivativethereof in preference to a second enantiomer of the enantiomeric mixtureor derivative thereof, and (ii) unreacted second enantiomer; andseparating the oligomer and unreacted second enantiomer from thereaction product and each other.
 73. A process as set forth in claim 72,wherein the first enantiomer is recovered from the separated oligomer byhydrolyzing the oligomer with acid and separating the α-amino acidenantiomer or derivative thereof from other hydrolyzates.
 74. A processas set forth in claim 73, wherein the α-amino acid enantiomer orderivative thereof is separated by subjection to enantioselectivechromatography.
 75. A process as set forth in claim 72, wherein theunreacted α-amino acid enantiomer or derivative thereof is recoveredfrom the reaction mixture by rotary evaporation.
 76. A process as setforth in claim 72, wherein the α-amino acid is selected from the groupconsisting of methionine and lysine.
 77. A process as set forth in claim72, wherein the α-hydroxy carboxylic acid comprises2-hydroxy-4-(methylthio)butyric acid or a derivative thereof.
 78. Aprocess for purifying an α-amino acid enantiomer or derivative thereofin an enantiomeric mixture, the process comprising: forming a reactionmixture comprising (i) an enantioselective enzyme and (ii) anenantiomeric mixture of an α-amino acid or derivative thereof; forming apeptide reaction product mixture comprising (i) an oligomer or aco-oligomer having a first enantiomer of the enantiomeric mixtureincorporated therein in preference to the second enantiomer of theenantiomeric mixture, and (ii) unreacted second enantiomer; andseparating the oligomer or co-oligomer and unreacted second enantiomerfrom the reaction product mixture and each other
 79. A process as setforth in claim 78, wherein the first enantiomer is recovered from theseparated oligomer or co-oligomer by hydrolyzing the oligomer orco-oligomer with acid and separating the α-amino acid enantiomer orderivative thereof from other hydrolyzates.
 80. A process as set forthin claim 79, wherein the first enantiomer or derivative thereof isseparated by subjection to enantioselective chromatography.
 81. Aprocess as set forth in claim 78, wherein the unreacted α-amino acidenantiomer or derivative thereof is recovered from the reaction mixtureby rotary evaporation.
 82. A process as set forth in claim 78 whereinthe α-hydroxy carboxylic acid derivative is a free acid, acid halide,amide, ester or anhydride.
 83. A process as set forth in claim 82wherein the α-amino acid derivative is a free acid, acid halide, amide,ester or anhydride.